Novel compositions from Nigella sativa

ABSTRACT

Described are four, novel supercritical fluid extracts of  Nigella sativa  seeds containing about 0.01 to about 40% (w/w) thymoquinone that can be produced using commercial-scale quantities of  N. sativa  seeds. These formulations provide antioxidant, thermogenic, anti-inflammatory and other biological activities qualitatively and quantitatively distinct from thymoquinone alone and are useful as dietary supplements or therapeutics for inflammation-related disorders.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. application Ser. No. 12/927,873filed Nov. 29, 2010, the entirety of which is incorporated herein byreference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to four novel compositions of Nigellasativa that function uniquely to reduce oxidative stress, inhibitsecretion of prostate specific antigen (PSA) from prostate cells,uncouple mitochondrial membrane potential, inhibit inducible nitricoxide synthase (iNOS) in inflamed fat and muscle tissue, modify fattyacid flux, activate myocyte AMP-activated protein Kinase (AMPK), andinhibit loss of transepithelial electrical resistance (TEER) in stressedintestinal epithelial cells in a manner that is unexpectedly bothquantitatively and qualitatively superior to thymoquinone (TQ), theputative active phytochemical of N. sativa (FIG. 1). These compositionswould be useful in the prevention or treatment of metabolic disorderssuch as adaptive thermogenesis, obesity, diabetes, and metabolicsyndrome as well as hyperlipidemia, hypertension and exercise recovery.The compositions would also be useful in the treatment or ameliorationof benign prostate hyperplasia.

2. Description of the Related Art

Nigella sativa, commonly known as black seed or black curcumin, istraditionally used in the Indian subcontinent, Arabian countries, andEurope for culinary and medicinal purposes as a natural remedy for anumber of illnesses and conditions that include asthma, hypertension,diabetes, inflammation, cough, bronchitis, headache, eczema, fever,dizziness and influenza. Much of the biological activity of the seeds isbelieved to be due to TQ, a component of the essential oil, which isalso present in the fixed oil.

The seeds of N. sativa as well as TQ are characterized by a very lowdegree of toxicity. Administration of either the seed, its extract orits oil has been shown not to induce significant toxicity or adverseeffects on liver or kidney functions even at extremely high doses [Ali BH, Blunden G. 2003. Pharmacological and toxicological properties ofNigella sativa. Phytother Res 17: 299-305]. Thus, N. sativa seed and TQpossess the necessary safety factor for commercialization in the dietarysupplement or pharmaceutical market.

TABLE 1 Chemical Content of Various Oil Fractions of N. sativa SeedsCONTENT [% SEED FRACTION COMPONENT [w/w] Fixed Oils 36 Non-fatcomponents Fatty acids, protein, thiamin, 58 ribovflavin, pyridoxine,niacin, folic acid, and calcium. Essential fatty acids Myristic acid(C14) 0.5 in fixed oil Palmitic acid (C16) 13.7 Palmitoleic acid (C16ω-9) 0.1 Stearic acid (C18) 2.6 Linoleic acid (C18 ω-6) 57.9 Linolenicacid (C18 ω-3 0.2 Arachidic acid (C20) 1.3 Essential oil components(0.5-1.5%): α-Pinene, camphene, β-pinene, sabinene, β-myrcene,α-terpinene, limonene, β-phellandrene, 1,8-cineole, γ-terpinene,p-cymene (7.1-15.5%), α-terpinolene, 2-heptanal, thujone,trans-sabinenehydrate, longipinene, camphor, linalool,cis-Sabinenehydrate, longifoline (1.0-8.0%), bornylacetate,2-undecanone, 4-terpineol (2.0-6.6%), borneol, carvone, thymoquinone(27-57%), 2-tridecanone, t-anethole (0.25-2.3%), p-cymene-8-ol,p-anisaldehyde, thymol and carvacrol (5.8-11.6%) (Burits, M.; Bucar, F.,Antioxidant activity of Nigella sativa essential oil. Phytother Res2000, 14 (5), 323-8).

As seen in Table 1, the TQ or dithymoquinone content of the essential(volatile) oil fraction is roughly 27-57 percent. The essential oilfraction, however, constitutes only one percent of the seed oils. Thus,TQ comprises only about 0.3 to 0.6% of the fixed oil fraction, the mostcommon commercially available product of N. sativa seeds.

Extraction Processes—

Traditional solvent extraction is time-consuming, requires multiplesteps, and consumes large amounts of organic solvents. The amount andthe price of organic solvent directly influences the total cost ofproducing an acceptable extract or product. Moreover, when the finalproduct is used as a food ingredient, it is absolutely necessary toremove all potentially toxic solvents.

Supercritical fluid extraction (SFE) has already proven itself as anattractive technique for selectively removing compounds from complexfood matrices. Extraction with liquid or supercritical CO₂ isessentially a simple concept, although specialized equipment andtechnically skilled operators are needed to bring concept to reality.CO₂ can exist in solid, liquid or gaseous phase, in common with allchemical substances. Furthermore, if the liquid phase is taken beyondthe so-called critical points of temperature and pressure, asupercritical fluid is formed, which in simple terms can be consideredas a dense gas1. Both liquid and supercritical CO₂ act effectively assolvents. While liquid CO₂ is excellent for dissolving relativelynon-polar, small molecules (liquid CO₂ can be compared to hexane in thisregard), supercritical CO₂ allows the extraction of larger and morepolar compounds. Thus, supercritical extraction has the potential forcreating novel extracts of commonly used herbs.

Supercritical CO₂ is pumped through the plant material in the extractioncolumns, where extraction of the desired plant components takes place.After passing through the expansion valve, the extract-laden CO₂ isdepressurised and the extract precipitates out of solution in theseparator. The gaseous CO₂ can be recycled for further extractions (FIG.2).

What sets liquid and supercritical CO₂ apart from other solvents such ashexane and ethanol are two key properties. Firstly, once the extractionhas been effected, the CO₂ solvent is released as a gas and recycled inthe process, so that a solvent-free extract is produce. This has twoimmediate benefits—the extract is free of all solvent residues, andimportantly so is the extracted material, which can then be further usedfor processing if required. Secondly, the solvating power of CO₂ can bemanipulated readily by altering temperature and pressure. This meansthat extraction can be highly selective and novel.

Obesity is a disease resulting from a prolonged positive imbalancebetween energy intake and energy expenditure. In 2000, an estimated30.5% of adults in the U.S. were obese (i.e. had a body mass index [BMI]greater than 30 kg/m²) and 15.5% of adolescents were overweight (BMI of25 to 30 kg/m²). Excess body weight is one of the most important riskfactors for all-cause morbidity and mortality. The likelihood ofdeveloping conditions such as type 2 diabetes, heart disease, cancer andosteoporosis of weight-bearing joints increases with body weight. Therapidly increasing world-wide incidence of obesity and its associationwith serious comorbid diseases means it is beginning to replaceundernutrition and infectious diseases as the most significantcontributor to ill health in the developed world.

It is now generally accepted that adipose tissue acts as an endocrineorgan producing a number of biologically active peptides with animportant role in the regulation of food intake, energy expenditure anda series of metabolic processes. Adipose tissue secretes a number ofbioactive peptides collectively termed adipokines. Through theirsecretory function, adipocytes lie at the heart of a complex networkcapable of influencing several physiological processes. Dysregulation ofadipokine production with alteration of adipocyte mass has beenimplicated in metabolic and cardiovascular complications of obesity. Inobese individuals, excessive production of acylation-stimulating protein(ASP), TNFα, IL-6 or resistin deteriorates insulin action in muscles andliver, while increased angiotensinogen and PAI-1 secretion favorshypertension and impaired fibrinolysis. Leptin regulates energy balanceand exerts an insulin-sensitizing effect. These beneficial effects arereduced in obesity due to leptin resistance. Adiponectin increasesinsulin action in muscles and liver and exerts an anti-atherogeniceffect. Further, adiponectin is the only known adipokine whosecirculating levels are decreased in the obese state. Thethiazolidinedione anti-diabetic drugs increase plasma adiponectin,supporting the idea that adipokine-targeted pharmacology represents apromising therapeutic approach to control type 2 diabetes andcardiovascular diseases in obesity.

Metabolism of white adipose tissue is involved in the control of bodyfat content, especially visceral adipose tissue. Adipose tissue plays acentral role in the control of energy homeostasis through the storageand turnover of triglycerides and through the secretion of factors thataffect satiety and fuel utilization. Mitochondrial remodeling andincreased energy expenditure in white fat may affect whole-body energyhomeostasis and insulin sensitivity [Wilson-Fritch L, Nicoloro S,Chouinard M, Lazar M A, Chui P C, et al. 2004. Mitochondrial remodelingin adipose tissue associated with obesity and treatment withrosiglitazone. J Clin Invest 114: 1281-9].

Oxidative Stress—

Current consensus is that hyperglycemia results in the production ofreactive oxygen (oxidative stress) and nitrogen species, which leads tooxidative myocardial injury. Alterations in myocardial structure andfunction occur in the late stage of diabetes. These chronic alterationsare believed to result from acute cardiac responses to suddenlyincreased glucose levels at the early stage of diabetes. Oxidativestress, induced by reactive oxygen and nitrogen species derived fromhyperglycemia, causes abnormal gene expression, altered signaltransduction, and the activation of pathways leading to programmedmyocardial cell deaths. The resulting myocardial cell loss thus plays acritical role in the development of diabetic cardiomyopathy.

Mitochondrial Uncoupling—

Controlling adiposity by targeted modulation of adipocyte mitochondrialmembrane potential could offer an attractive alternative to currentdietary approaches. It has recently been reported that forced uncouplingprotein 1 (UCP1) expression in white adipocytes derived from a murine(3T3-L1) preadipocyte cell line reduced the total lipid accumulation byapproximately 30% without affecting other adipocyte markers, such ascytosolic glycerol-3-phosphate dehydrogenase activity and leptinproduction. The expression of UCP1 also decreased glycerol output andincreased glucose uptake, lactate output, and the sensitivity ofcellular ATP content to nutrient removal [Si Y, Palani S, Jayaraman A,Lee K. 2007. Effects of forced uncoupling protein 1 expression in 3T3-L1cells on mitochondrial function and lipid metabolism. J Lipid Res 48:826-36]. These results suggest that the targeting reduction inintracellular lipid of adipocytes by uncoupling mitochondrial membranepotential represents a feasible mechanism for identification ofanti-obesity molecules. Nevertheless, the putative role of variousmitochondrial protonophores in white fat cells in the control ofadiposity remains to be clarified.

Thermogenesis—

Thermogenesis or uncoupling of mitochondrial membrane potential may beactivated both indirectly and directly. Indirect activation occursthrough β3AR and β3 agonists (β3AA). In the early 1980s, an “atypical”beta-adrenergic receptor was discovered and subsequently called β3AR.Further clinical testing will be necessary, using compounds withimproved oral bioavailability and potency, to help assess the physiologyof the β3AR in humans and its attractiveness as a potential therapeuticfor the treatment of type 2 diabetes and obesity [de Souza C J, Burkey BF. 2001. Beta 3-adrenoceptor agonists as anti-diabetic and anti-obesitydrugs in humans. Curr Pharm Des 7: 1433-49].

Adaptive Thermogenesis—

Adaptive thermogenesis represents the decrease in energy expenditure(EE) beyond what could be predicted from the changes in fat mass orfat-free mass under conditions of standardized physical activity inresponse to a decrease in energy intake. Thus there exists the potentialof adaptive thermogenesis to impede obesity treatment on a short- andlong-term basis, at least in some individuals. In some cases, theadaptive decrease in thermogenesis was shown to be significantly relatedto a single cycle of body weight loss and regain, an increase in plasmaorganochlorine concentration following weight loss. This suggests thatenergy metabolism might be sensitive to stimuli of differentphysiological nature and that adaptive thermogenesis could bequantitatively more important than what is generally perceived by healthprofessionals and nutrition specialists. However, from a clinical pointof view, several issues remain to be investigated in order to moreclearly identify adaptive thermogenesis determining factors and todevelop strategies to cope with them. Along these lines, it is concludedthat unsuccessful weight loss interventions and reduced body weightmaintenance could be partly due, in some vulnerable individuals, to theadaptive thermogenesis, which is multicausal, quantitativelysignificant, and has the capacity to compensate for a given prescribedenergy deficit, possibly going beyond any good compliance of somepatients.

Additional approaches to increasing thermogenesis appear necessary toaffect sustained weight loss in obese subjects. One of these approacheswith demonstrated proof-of-concept in humans is direct, chemicalstimulation of thermogenesis through chemical uncoupling ofmitochondrial membrane potential using 2,4-dinitrophenol (DNP). Doublingmetabolic rate by selectively and modestly uncoupling adipocytethermogenesis should produce few adverse side-effects as this level ofincrease would only be equivalent to mild exercise. DNP is alipid—soluble, weak acid that acts as a protonophore because it cancross membranes protonated, lose its proton and return as the anion,then reprotonate and repeat the cycle. In this way, it increases thebasal proton conductance of mitochondria and uncouples oxidativephosphorylation. The overall result is a decrease in ATP formation foran equivalent amount of oxidation.

Inducible Nitric Oxide Synthase—

Obesity leading to insulin resistance is a major causative factor fortype 2 diabetes and is associated with increased risk of cardiovasculardisease. Despite intense investigation for a number of years, molecularmechanisms underlying insulin resistance remain to be determined.Recently, chronic inflammation has been highlighted as a culprit forobesity-induced insulin resistance. Nonetheless, upstream regulators anddownstream effectors of chronic inflammation in insulin resistanceremain unclarified. Inducible nitric oxide synthase (iNOS), a mediatorof inflammation, has emerged as an important player in insulinresistance. Obesity is associated with increased iNOS expression ininsulin-sensitive tissues in rodents and humans. Inhibition of iNOSameliorates obesity-induced insulin resistance. However, molecularmechanisms by which iNOS mediates insulin resistance via nitric oxide(NO) biosynthesis remain largely unknown.

NO is a critically important signaling molecule, controlling a widerange of pathways and biological processes. Highly reactive nitric oxidemediates its function through reaction with different molecules directlyor indirectly. One of these modifications is the S-nitrosylation ofcysteine residues in proteins. S-nitrosylation is emerging as animportant redox signaling mechanism and has been found to regulate abroad range of biologic, physiologic and cellular functions [Hausladen,A., and Stamler, J. S, Nitrosative stress. Methods Enzymol 1999, 300,389-95].

Protein S-nitrosylation, a covalent attachment of NO moiety to thiolsulfhydryls, has emerged as a major mediator of a broad array of NOactions. S-nitrosylation is elevated in patients with type 2 diabetes,and increased S-nitrosylation of insulin signaling molecules, includinginsulin receptor, insulin receptor substrate-1, and Akt/PKB, has beenshown in skeletal muscle of obese, diabetic mice. Akt/PKB is reversiblyinactivated by S-nitrosylation. Based on these findings, S-nitrosylationhas recently been proposed to play an important role in the pathogenesisof insulin resistance [Kaneki, M., Shimizu, N., Yamada, D., and Chang,K. Nitrosative stress and pathogenesis of insulin resistance. AntioxidRedox Signal 2007, 9, 319-29].

Moreover iNOS expression is increased in skeletal muscle of diabetic(ob/ob) mice compared with lean wild-type mice. iNOS gene disruption ortreatment with iNOS inhibitor ameliorates depressed IRS-1 expression inskeletal muscle of diabetic (ob/ob) mice. These findings indicate thatiNOS reduces IRS-1 expression in skeletal muscle via proteasome-mediateddegradation and thereby may contribute to obesity-related insulinresistance [Sugita, H., Fujimoto, M., Yasukawa, T., Shimizu, N., Sugita,M., Yasuhara, S., Martyn, J. A., and Kaneki, M. Inducible nitric-oxidesynthase and NO donor induce insulin receptor substrate-1 degradation inskeletal muscle cells. J Biol Chem 2005, 280, 14203-11].

Improvements in the treatment of noncardiac complications from diabeteshave resulted in heart disease becoming a leading cause of death indiabetic patients. Pathogenesis of diabetic cardiomyopathy (DCM) is acomplicated and chronic process that is secondary to acute cardiacresponses to diabetes. One of the acute responses is cardiac cell deaththat plays a critical role in the initiation and development of DCM.Besides hyperglycemia, inflammatory response in the diabetic heart isalso a major cause for cardiac cell death. Diabetes or obesity oftencauses systemic and cardiac increases in tumor necrosis factor-alpha(TNFα), interleukin-18 and PAI-1. However, how these cytokines causecardiac cell death remains unclear. It has been considered to relate tooxidative and/or nitrosative stress. Cardiac cell death is induced bythe inflammatory cytokines that are increased in response to diabetes.Inflammatory cytokine-induced cardiac cell death is mediated byoxidative stress and is also the major initiator for DCM development[Wang, Y. H., and Cai, L. Diabetes/obesity-related inflammation, cardiaccell death and cardiomyopathy. Zhong Nan Da Xue Xue Bao Yi Xue Ban 2006,31, 814-8].

AMP-Activated Protein Kinase—

The 5′-AMP-activated protein kinase (AMPK) functions as an intracellularfuel sensor that affects metabolism and gene expression in humans androdents. AMPK has been described as an integrator of regulatory signalsmonitoring systemic and cellular energy status. Recently, it has beenproposed that AMPK could provide a link in metabolic defects underlyingprogression to the metabolic syndrome. AMPK is a heterotrimeric enzymecomplex consisting of a catalytic subunit alpha and two regulatorysubunits beta and gamma. Rising AMP and falling ATP activate AMPK. AMPactivates the system by binding to the gamma subunit that triggersphosphorylation of the catalytic alpha subunit by the upstream kinasesLKB1 and CaMKKbeta (calmodulin-dependent protein kinase kinase). TheAMPK system is a regulator of energy balance that, once activated by lowenergy status, switches on ATP-producing catabolic pathways (such asfatty acid oxidation and glycolysis), and switches off ATP-consuminganabolic pathways (such as lipogenesis), both by short-term effect onphosphorylation of regulatory proteins and by long-term effect on geneexpression (FIG. 3).

As well as acting at the level of the individual cell, the system alsoregulates food intake and energy expenditure at the whole body level, inparticular by mediating the effects of insulin sensitizing adipokinesleptin and adiponectin. AMPK is robustly activated during skeletalmuscle contraction and myocardial ischemia playing a role in glucosetransport and fatty acid oxidation. In liver, activation of AMPK resultsin enhanced fatty acid oxidation as well as decreased glucose production[Viollet, B., Mounier, R., Leclerc, J., Yazigi, A., Foretz, M., andAndreelli, F. Targeting AMP-activated protein kinase as a noveltherapeutic approach for the treatment of metabolic disorders. DiabetesMetab 2007, 33, 395-402]. The net effect of AMPK activation isstimulation of hepatic fatty acid oxidation and ketogenesis, inhibitionof cholesterol synthesis, lipogenesis, and triglyceride synthesis,inhibition of adipocyte lipolysis and lipogenesis, stimulation ofskeletal muscle fatty acid oxidation and muscle glucose uptake, andmodulation of insulin secretion by pancreatic beta-cells.

5-Aminoimidazole-4-carboxamide ribonucleoside (AICAR) represents auseful tool for identifying new target pathways and processes regulatedby the AMPK protein kinase cascade. Incubation of rat hepatocytes withAICAR results in accumulation of the monophosphorylated derivative(5-aminoimidaz-ole-4-carboxamide ribonucleoside; ZMP) within the cell.ZMP mimics both activating effects of AMP on AMPK, i.e. directallosteric activation and promotion of phosphorylation by AMPK kinase.Unlike existing methods for activating AMPK in intact cells (e.g.fructose, heat shock), AICAR does not perturb the cellular contents ofATP, ADP or AMP. Incubation of hepatocytes with AICAR activates AMPK dueto increased phosphorylation, causes phosphorylation and inactivation ofa known target for AMPK (3-hydroxy-3-methylglutaryl-CoA reductase), andalmost total cessation of two of the known target pathways, i.e. fattyacid and sterol synthesis. Incubation of isolated adipocytes with AICARantagonizes isoprenaline-induced lipolysis. This provides directevidence that the inhibition by AMPK of activation of hormone-sensitivelipase by cyclic-AMP-dependent protein kinase, previously demonstratedin cell-free assays, also operates in intact cells.

AMPK also regulates food intake and energy expenditure at the whole bodylevel, in particular by mediating the effects of insulin sensitizingadipokines leptin and adiponectin. AMPK is robustly activated duringskeletal muscle contraction and myocardial ischemia playing a role inglucose transport and fatty acid oxidation.

Additional approaches to affect sustained weight loss in obese subjectsrepresent a critical need. Further, compounds or formulations thatsafely and effectively activate AMPK may function to stimulate hepaticfatty acid oxidation and ketogenesis, inhibit cholesterol synthesis,lipogenesis, and triglyceride synthesis, inhibit adipocyte lipolysis andlipogenesis, stimulate of skeletal muscle fatty acid oxidation andmuscle glucose uptake, and modulate insulin secretion by pancreaticbeta-cells.

Inflammatory Bowel Disease—

Each year inflammatory bowel diseases, such as Crohn's disease andulcerative colitis, afflict more than one million people in the UnitedStates [Baumgart D C, Bernstein C N, Abbas Z, et al. IBD Around theworld: Comparing the epidemiology, diagnosis, and treatment: Proceedingsof the World Digestive Health Day 2010—Inflammatory bowel disease taskforce meeting. Inflamm Bowel Dis 2010]. The healthy gastrointestinaltract absorbs only the small molecules like those that are product ofcomplete digestion. These molecules are the amino acids, simple sugars,fatty acids, vitamins, and minerals that the body requires for all theprocesses of life to function properly. The intestines, small intestinein particular, only allow these substances to enter the body due to thefact that the cells that make up the intestinal wall are tightly packedtogether. The intestines also contain special proteins called ‘carrierproteins’ that are responsible for binding to certain nutrients andtransporting them through the intestinal wall and into the bloodstream.

Leaky Gut Syndrome (LGS) is common parlance for the disruption of theintestinal membrane integrity as a result of oxidative stressors orpro-inflammatory mediators, which compromise the ability of theintestinal wall to keep out large and undesirable molecules. Hence thename, as substances that are normally kept outside the body and withinthe intestines, are “leaking” across the intestinal wall and into thebody as a whole. This happens when the spaces between the cells of theintestinal wall become enlarged for various reasons and allow larger,less digested particles and toxins to pass through—causing LGS. The bodythen recognizes these particles as foreign “invaders,” and the immunesystem attempts to fight them off—which can set the stage for variousautoimmune disorders.

This disruption of intestinal membrane integrity is applicable to thepathognomic impacts of asthma, arthritis, food allergies, ulcers,Crohn's disease, ulcerative colitis, celiac disease, autoimmunediseases, alcoholism, chronic fatigue, joint pain, migraines, diarrhea,parasitic infections, dysbiosis, candidiasis, multiple sclerosis,diabetes, multiple sclerosis, vasculitis, Addison's disease, lupus,thyroiditis, and fibromyalgia.

Novel, supercritical CO₂ extracts of N. sativa are described thatcontain from about 0.01 to about 39% (w/w) TQ and unexpectedly exhibitchemical and biological activities in vitro and clinically that differboth qualitatively and quantitatively from TQ, the putative activecomponent of N. sativa seed extracts.

To date no process for supercritical extraction of N. sativa has beendescribed that is useful for commercial quantities of ground seed. It iswell known in the art, that changes in scale will profoundly affect thequality and quantity of the extract produced. The procedure describedherein can be used in the extraction of commercial quantities of N.sativa seed in amounts greater than about 10 kg.

SUMMARY OF THE INVENTION

The present invention provides a method of making four commercial-scalecompositions enriched in essential oils from seeds of Nigella sativacomprising the steps of: (i) grinding and sieving the seed of Nigellasativa to a fine powder about 20 to 30 mesh; (ii) extracting quantitiesof the ground seeds in amounts greater than 1 kg with supercritical CO₂at about 140 bar, 50° C. for 30 minutes and collecting the fractionobtained; (iii) continuing the extraction at about 140 bar, 50° C. for120 minutes and collecting the fraction obtained; (iv) increasing thepressure to about 300 bar, temperature to 60° C. and collection time toan additional 180 minutes and collecting the fraction obtained; and (v)formulating a fourth composition by mixing the extracted, spent N.sativa powdered seeds with the essential oil fraction collected at about300 bar, 60° C. and 180 minutes in a ratio of about 24:1.

The present invention further provides four novel, supercritical CO₂extracts of ground seeds obtained from N. sativa produced on acommercial scale. The novel compositions thus produced can be used (i)to reduce oxidative stress, (ii) to inhibit excessive prostate specificantigen secretion from precancerous prostate cells, (iii) to uncouplemitochondrial membrane potential in adipocytes, (iv) to inhibit iNOSmediated NO production in adipocytes and myocytes from multiple cytokinestimulation simultaneously, (v) to increase lipolysis in adipocytes andmyocytes, (v) to activate AMPK in myocytes, (vi) to overcome adaptivethermogenesis in humans resulting in more effective weight loss withexercise, (vii) to reduce inflammatory and oxidative loss of intestinalmembrane integrity, and (viii) to attenuate t10-CLA mediated proinflammatory effects on adipocyte secretion of IL-6 and adiponectin.

The present invention further provides a method of treating diseases orpathologies related to oxidative stress, inflammation, metabolicsyndrome, type 1 or type 2 diabetes, and obesity in an animal comprisingadministering to an animal exhibiting signs, signalments, or symptoms ofthe pathology or disease an effective amount of the supercriticalextract and continuing the administration of the composition until thesigns, signalments or symptoms are reduced.

The present invention relates to the unexpected discovery that certainsuper critical fluid CO₂ extracts of N. sativa decrease mitochondrialmembrane potential in adipocytes implying decreased ATP synthesis andincreased thermogenesis. The invention provides methods for modifyingadipocyte or myocyte physiology in a subject, comprising administeringto the subject a pharmaceutical composition of a supercritical fluidextract of N. sativa or mixtures thereof. Preferred embodiments providecompositions and methods for enhancing adipocyte thermogenesis ordecreasing oxidative stress utilizing supercritical fluid extracts of N.sativa.

Further, the present invention relates to the unexpected discovery thatcertain super critical fluid extracts of N. sativa inhibit iNOS-mediatedNO biosynthesis as a result of the action of multiple external stimulito adipocytes or myocytes implying inhibition of protein nitrosylation.

Additionally, the present invention relates to the unexpected discoverythat certain super critical fluid extracts of N. sativa dramaticallyactivate AMPK implying stimulation of hepatic fatty acid oxidation andketogenesis, inhibition of cholesterol synthesis, lipogenesis, andtriglyceride synthesis, inhibition of adipocyte lipolysis andlipogenesis, stimulation of skeletal muscle fatty acid oxidation andmuscle glucose uptake, and modulation of insulin secretion by pancreaticbeta-cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is the structure of [A] thymoquinone and [B] dithymoquinone.

FIG. 2 is a schematic diagram of the supercritical CO₂ extractioncircuit used in Example 1.

FIG. 2 depicts the role of AMPK in regulating energy balance at thewhole-body level. Arrows indicate positive effects, and bars indicatenegative effects. FA=fatty acid.

FIG. 4 describes the unique fractions obtained under various conditionsof supercritical CO₂ extraction of N. sativa powdered seeds.

FIG. 5 depicts the relative inhibition and stimulation, respectively, ofprostate specific antigen by N. sativa supercritical fraction NS9 andpure TQ adjusted for viable LNCaP prostate cells.

FIG. 6 depicts the dose-related decrease and dose-related increase,respectively, of prostate specific antigen by N. sativa supercriticalfractions NS8 and NS9 adjusted for viable LNCaP cells.

FIG. 7 depicts the relative inhibition and stimulation, respectively, ofprostate specific antigen by N. sativa supercritical fraction NS/Spent#2and Spent#1 adjusted for viable LNCaP cells.

FIG. 8 depicts the relative FC-1 monomer/aggregate fluorescence in3T3-L1 adipocytes treated with 500 μM dinitrophenol or test materialsTQ, NS8, NS9 or NS10 dosed at 25 μg/mL.

FIG. 9 depicts the relative free fatty acid release from 3T3-L1adipoctyes treated with 82 μg/mL forskolin or test materials TQ, NS8,NS9, or NS 10 dosed at 5 μg/mL.

FIG. 10 depicts the relative free fatty acid release from C2C12 myocytestreated with materials NS8, NS9, or NS10 dosed at 5 or 2.5 μg/mL.

FIG. 11 depicts the relative increase in pAMPK in C2C12 myocytes treatedwith 1 mM AICAR and test materials NS8, NS9, or NS10 dosed at 25 μg/mL.

FIG. 12 depicts the change in body weight of a 61-year old male, who hadpreviously exhibited adaptive thermogenesis, during a week followingdaily dosing of 40 mg NS9/kg-day. Error bars are the 99% confidenceinterval computed from the previous six-month variation in day-to-daybody weight for am and pm weighing.

DETAILED DESCRIPTION OF THE INVENTION

The compositions of the invention includes four, unique supercriticalfluid extracts of N. sativa containing concentrations of the TQ markercompound ranging from about 0.01 to about 40%. These compositions may beused for their antioxidant or anti-inflammatory properties. Theresulting compositions can be consumed as a dietary supplement toaddress the risk factors associated with oxidative stress, benignprostate hyperplasia, obesity, metabolic syndrome, diabetes, increasingexercise endurance or other inflammatory-based pathologies.

The patents, published applications, and scientific literature referredto herein establish the knowledge of those with skill in the art and arehereby incorporated by reference in their entirety to the same extent asif each was specifically and individually indicated to be incorporatedby reference. Any conflict between any reference cited herein and thespecific teachings of this specification shall be resolved in favor ofthe latter. Likewise, any conflict between an art-understood definitionof a word or phrase and a definition of the word or phrase asspecifically taught in this specification shall be resolved in favor ofthe latter.

Technical and scientific terms used herein have the meaning commonlyunderstood by one of skill in the art to which the present inventionpertains, unless otherwise defined. Reference is made herein to variousmethodologies and materials known to those of skill in the art. Standardreference works setting forth the general principles of recombinant DNAtechnology include Sambrook et al., Molecular Cloning: A LaboratoryManual, 2nd Ed., Cold Spring Harbor Laboratory Press, New York (1989);Kaufman et al., Eds., Handbook of Molecular and Cellular Methods inBiology in Medicine, CRC Press, Boca Raton (1995); McPherson, Ed.,Directed Mutagenesis: A Practical Approach, IRL Press, Oxford (1991).Standard reference works setting forth general definitions of medicalterms and the general principles of pharmacology, respectively, includeStedman's Medical Dictionary [26^(th) edition] and Goodman and Gilman'sThe Pharmacological Basis of Therapeutics, 11th Ed., McGraw HillCompanies Inc., New York (2006).

In the specification and the appended claims, the singular forms includeplural referents unless the context clearly dictates otherwise. As usedin this specification, the singular forms “a,” “an” and “the”specifically also encompass the plural forms of the terms to which theyrefer, unless the content clearly dictates otherwise. Additionally, asused herein, unless specifically indicated otherwise, the word “or” isused in the “inclusive” sense of “and/or” and not the “exclusive” senseof “either/or.” The term “about” is used herein to mean approximately,in the region of, roughly, or around. When the term “about” is used inconjunction with a numerical range, it modifies that range by extendingthe boundaries above and below the numerical values set forth. Ingeneral, the term “about” is used herein to modify a numerical valueabove and below the stated value by a variance of 20%.

As used herein, the recitation of a numerical range for a variable isintended to convey that the invention may be practiced with the variableequal to any of the values within that range. Thus, for a variable thatis inherently discrete, the variable can be equal to any integer valueof the numerical range, including the end-points of the range.Similarly, for a variable that is inherently continuous, the variablecan be equal to any real value of the numerical range, including theend-points of the range. As an example, a variable that is described ashaving values between 0 and 2 can be 0, 1 or 2 for variables that areinherently discrete, and can be 0.0, 0.1, 0.01, 0.001, or any other realvalue for variables that are inherently continuous.

As used in this specification, whether in a transitional phrase or inthe body of the claim, the terms “comprise(s)” and “comprising” are tobe interpreted as having an open-ended meaning. That is, the terms areto be interpreted synonymously with the phrases “having at least” or“including at least”. When used in the context of a process, the term“comprising” means that the process includes at least the recited steps,but may include additional steps. When used in the context of a compoundor composition, the term “comprising” means that the compound orcomposition includes at least the recited features or compounds, but mayalso include additional features or compounds.

Reference is made hereinafter in detail to specific embodiments of theinvention. While the invention will be described in conjunction withthese specific embodiments, it will be understood that it is notintended to limit the invention to such specific embodiments. On thecontrary, it is intended to cover alternatives, modifications, andequivalents as may be included within the spirit and scope of theinvention as defined by the appended claims. In the followingdescription, numerous specific details are set forth in order to providea thorough understanding of the present invention. The present inventionmay be practiced without some or all of these specific details. In otherinstances, well known process operations have not been described indetail, in order not to unnecessarily obscure the present invention.

Any suitable materials and/or methods known to those of skill can beutilized in carrying out the present invention. However, preferredmaterials and methods are described. Materials, reagents and the like towhich reference are made in the following description and examples areobtainable from commercial sources, unless otherwise noted.

The term “treat” and its verbal variants refer to palliation oramelioration of an undesirable physiological state. Thus, for example,where the physiological state is poor glucose tolerance, “treatment”refers to improving the glucose tolerance of a treated subject. Asanother example, where the physiological state is obesity, the term“treatment” refers to reducing the body fat mass, improving the bodymass or improving the body fat ratio of a subject. Treatment of diabetesmeans improvement of blood glucose control. Treatment of inflammatorydiseases means reducing the inflammatory response either systemically orlocally within the body. Treatment of osteoporosis means an increase inthe density of bone mineralization or a favorable change in metabolic orsystemic markers of bone mineralization. The person skilled in the artwill recognize that treatment may, but need not always, includeremission or cure.

The term “prevent” and its variants refer to prophylaxis against aparticular undesirable physiological condition. The prophylaxis may bepartial or complete. Partial prophylaxis may result in the delayed onsetof a physiological condition. The person skilled in the art willrecognize the desirability of delaying onset of a physiologicalcondition, and will know to administer the compositions of the inventionto subjects who are at risk for certain physiological conditions inorder to delay the onset of those conditions. For example, the personskilled in the art will recognize that obese subjects are at elevatedrisk for coronary artery disease. Thus, the person skilled in the artwill administer compositions of the invention in order to increaseinsulin sensitivity in an obese, whereby the onset of diabetes mellitusor dyslipemia may be prevented entirely or delayed.

As used herein “adaptive thermogenesis” represents the decrease inenergy expenditure beyond what could be predicted from the changes infat mass or fat-free mass under conditions of standardized physicalactivity in response to a decrease in energy intake.

As used herein the term “oxidative stress” is used to describe theeffect of oxidation in which an abnormal level of reactive oxygenspecies (ROS), such as the free radicals (e.g. hydroxyl, nitric acid,superoxide) or the non-radicals (e.g. hydrogen peroxide, lipid peroxide)lead to damage (called oxidative damage) to specific molecules withconsequential injury to cells or tissue. Increased production of ROSoccurs as a result of fungal or viral infection, inflammation, ageing,UV radiation, pollution, excessive alcohol consumption, cigarettesmoking, etc. Removal or neutralization of ROS is achieved withantioxidants, endogenous (e.g. catalase, glutathione, superoxidedismutase) or exogenous (e.g. vitamins A, C, E, bioflavonoids,carotenoids). Oxidative damage to the eye, particularly the retina andthe lens, is a contributing factor to age-related macular degenerationand cataract.

All forms of life maintain a reducing environment within their cells.This reducing environment is preserved by enzymes that maintain thereduced state through a constant input of metabolic energy. Disturbancesin this normal redox state can cause toxic effects through theproduction of peroxides and free radicals that damage all components ofthe cell, including proteins, lipids and DNA.

In humans, oxidative stress is involved in the etiology of manydiseases, such as atherosclerosis, type 1 and type 2 diabetes,Parkinson's disease, cardiac arrest, myocardial infarction, Alzheimer'sdisease, Fragile X syndrome and chronic fatigue syndrome. Short-termoxidative stress, however, may also be important in prevention of agingby induction of a process named mitohormesis. ROS can be beneficial, asthey are used by the immune system as a way to attack and kill invadingpathogens. ROS are also used in cell signaling. This is dubbed redoxsignaling and is a critical component of such pathognomic conditions asobesity, metabolic syndrome, colitis, irritable bowel syndrome andadaptive thermogenesis.

The methods of the present invention are intended for use with anysubject that may experience the benefits of the methods of theinvention. Thus, in accordance with the invention, “subjects” includehumans as well as non-human subject, particularly domesticated animals.It will be understood that the subject to which a compound of theinvention is administered need not suffer from a specific traumaticstate. Indeed, the compounds of the invention may be administeredprophylactically, prior to any development of symptoms. The term“therapeutic,” “therapeutically,” and permutations of these terms areused to encompass therapeutic, palliative as well as prophylactic uses.

As used herein, the term “solvent” refers to a liquid of gaseous,aqueous or organic nature possessing the necessary characteristics toextract solid material from the hop plant product. Examples of solventswould include, but not limited to, water, steam, superheated water,methanol, ethanol, hexane, chloroform, liquid CO₂, liquid N₂, propane,or any combinations of such materials.

As used herein, the term “CO₂ extract” refers to the solid materialresulting from exposing more than 10 kg powdered N. sativa seeds to aliquid or supercritical CO₂ preparation followed by subsequent removalof the CO₂.

As used herein, “decreased secretion or biosynthesis,” means to decreaseby at least 3%, the rate of secretion or amount of biosynthesis of thereferent compound. The invention further provides a method of decreasingadipocyte or myocyte concentrations of inflammatory mediators in asubject, comprising administering to the subject an amount of thecomposition sufficient to decrease NO secretion from adipocytes ormyocytes in the subject. In general, a decrease in adipocyte or myocyteNO secretion or biosynthesis will result in improvements in suchconditions as obesity, metabolic syndrome, colitis, irritable bowelsyndrome and adaptive thermogenesis.

As used herein, “linear inhibitory effect” refers to a linear decreasein secretion or biosynthesis resulting from all concentrations of theinhibiting material over a dose-response curve. For example, inhibitionat low concentrations followed by a failure of inhibition or increasedsecretion at higher concentrations represents a lack of a linearinhibitory effect.

As used herein, “Leaky Gut Syndrome (LGS)” is an increase inpermeability of the intestinal mucosa to luminal macromolecules,antigens and toxins associated with inflammatory degenerative and/oratrophic mucosal damage. LGS can lead to any number of seeminglyunrelated symptoms affecting every organ system in the body. LGS hasalso been linked with having a causative role in a large number ofdistinct illnesses. Many of these are autoimmune diseases, which meansthe immune system attacks the body's own cells. LGS plays a role inthese types of illness because it increases immune reactions to foodparticles and then cross reactivity may occur meaning that the immunesystem attacks body tissues that are chemically similar to the foods towhich it has become sensitized. A sampling of the many diseases in whichleaky gut syndrome may have a role includes: rheumatoid arthritis,osteoarthritis, asthma, multiple sclerosis, vasculitis, Crohn's Disease,colitis, Addison's Disease, lupus, thyroiditis, chronic fatiguesyndrome, and fibromyalgia.

As used herein, the term “CLA isomers” refers to fatty acids with thesame 18-carbon, polyunsaturated structure. In the case of CLA, eachisomer is derived from the 18-carbon essential polyunsaturated fatlinoleic acid (18:2n-6), which has two cis-double bonds at carbons 9 and12. Cis9-CLA has been shown to regulate adiposity in animals and humans.The trans10-CLA isomer (t10-CLA), however, is associated withhyperglycemia, insulin resistance and dyslipidemia as well as elevatedlevels of inflammatory prostaglandins and cytokines. These stressors canimpair the adipocyte's ability to synthesize or store fatty acids astriglycerides, causing lipids to accumulate in hepatocytes and myocytesand resulting in steatosis and insulin resistance, respectively. Theseissues raise concern about the safe and effective use of supplementscontaining t10-CLA as a dietary strategy for weight loss.

In some aspects the compositions further comprise a pharmaceuticallyacceptable excipient where the pharmaceutically acceptable excipient isselected from the group consisting of coatings, isotonic and absorptiondelaying agents, binders, adhesives, lubricants, disintergrants,coloring agents, flavoring agents, sweetening agents, absorbants,detergents, and emulsifying agents. In yet further aspects, thecomposition additionally comprises one or more members selected from thegroup consisting of antioxidants, vitamins, minerals, proteins, fats,and carbohydrates.

The term “therapeutically effective amount” is used to denote treatmentsat dosages effective to achieve the therapeutic result sought.Furthermore, one of skill will appreciate that the therapeuticallyeffective amount of the compound of the invention may be lowered orincreased by fine-tuning and/or by administering more than one compoundof the invention, or by administering a compound of the invention withanother compound. See, for example, Meiner, C. L., “Clinical Trials:Design, Conduct, and Analysis,” Monographs in Epidemiology andBiostatistics, Vol. 8 Oxford University Press, USA (1986). The inventiontherefore provides a method to tailor the administration/treatment tothe particular exigencies specific to a given mammal. As illustrated inthe following examples, therapeutically effective amounts may be easilydetermined, for example, empirically by starting at relatively lowamounts and by step-wise increments with concurrent evaluation ofbeneficial effect.

As used herein, “more effectively” is used to describe relativebiological responses of compounds or formulations wherein the responseelicited by one formulation is greater per unit dose than the other.

The term “pharmaceutically acceptable” is used in the sense of beingcompatible with the other ingredients of the compositions and notdeleterious to the recipient thereof.

As used herein, “compounds” may be identified either by their chemicalstructure, chemical name, or common name. When the chemical structureand chemical or common name conflict, the chemical structure isdeterminative of the identity of the compound. The compounds describedherein may contain one or more chiral centers and/or double bonds andtherefore, may exist as stereoisomers, such as double-bond isomers(i.e., geometric isomers), enantiomers or diastereomers. Accordingly,the chemical structures depicted herein encompass all possibleenantiomers and stereoisomers of the illustrated or identified compoundsincluding the stereoisomerically pure form (e.g., geometrically pure,enantiomerically pure or diastereomerically pure) and enantiomeric andstereoisomeric mixtures. Enantiomeric and stereoisomeric mixtures can beresolved into their component enantiomers or stereoisomers usingseparation techniques or chiral synthesis techniques well known to theskilled artisan. The compounds may also exist in several tautomericforms including the enol form, the keto form and mixtures thereof.Accordingly, the chemical structures depicted herein encompass allpossible tautomeric forms of the illustrated or identified compounds.The compounds described also encompass isotopically labeled compoundswhere one or more atoms have an atomic mass different from the atomicmass conventionally found in nature. Examples of isotopes that may beincorporated into the compounds of the invention include, but are notlimited to, ²H, ³H, ¹³C, ¹⁴C, ¹⁵N, ¹⁸O, ¹⁷O, etc. Compounds may exist inunsolvated forms as well as solvated forms, including hydrated forms andas N-oxides. In general, compounds may be hydrated, solvated orN-oxides. Certain compounds may exist in multiple crystalline oramorphous forms. Also contemplated within the scope of the invention arecongeners, analogs, hydrolysis products, metabolites and precursor orprodrugs of the compound. In general, all physical forms are equivalentfor the uses contemplated herein and are intended to be within the scopeof the present invention.

The compounds according to the invention are optionally formulated in apharmaceutically acceptable vehicle with any of the well-knownpharmaceutically acceptable carriers, including diluents and excipients(see Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, MackPublishing Co., Easton, Pa. 1990 and Remington: The Science and Practiceof Pharmacy, Lippincott, Williams & Wilkins, 1995). While the type ofpharmaceutically acceptable carrier/vehicle employed in generating thecompositions of the invention will vary depending upon the mode ofadministration of the composition to a mammal, generallypharmaceutically acceptable carriers are physiologically inert andnon-toxic. Formulations of compositions according to the invention maycontain more than one type of compound of the invention), as well anyother pharmacologically active ingredient useful for the treatment ofthe symptom/condition being treated.

The compounds of the present invention may be provided in apharmaceutically acceptable vehicle using formulation methods known tothose of ordinary skill in the art. The compositions of the inventioncan be administered by standard routes. The compositions of theinvention include those suitable for oral, inhalation, rectal,ophthalmic (including intravitreal or intracameral), nasal, topical(including buccal and sublingual), vaginal, or parenteral (includingsubcutaneous, intramuscular, intravenous, intradermal, andintratracheal). In addition, polymers may be added according to standardmethodologies in the art for sustained release of a given compound.

It is contemplated within the scope of the invention that compositionsused to treat a disease or condition will use a pharmaceutical gradecompound and that the composition will further comprise apharmaceutically acceptable carrier. It is further contemplated thatthese compositions of the invention may be prepared in unit dosage formsappropriate to both the route of administration and the disease andpatient to be treated. The compositions may conveniently be presented indosage unit form be prepared by any of the methods well known in the artof pharmacy. All methods include the step of bringing the activeingredient into association with the vehicle that constitutes one ormore auxiliary constituents. In general, the compositions are preparedby uniformly and intimately bringing the active ingredient intoassociation with a liquid vehicle or a finely divided solid vehicle orboth, and then, if necessary, shaping the product into the desiredcomposition.

The term “dosage unit” is understood to mean a unitary, i.e. a singledose which is capable of being administered to a patient, and which maybe readily handled and packed, remaining as a physically and chemicallystable unit dose comprising either the active ingredient as such or amixture of it with solid or liquid pharmaceutical vehicle materials.

Compositions suitable for oral administration may be in the form ofdiscrete units as capsules, sachets, tablets, soft gels or lozenges,each containing a predetermined amount of the active ingredient; in theform of a powder or granules; in the form of a solution or a suspensionin an aqueous liquid or non-aqueous liquid, such as ethanol or glycerol;or in the form of an oil-in-water emulsion or a water-in-oil emulsion.Such oils may be edible oils, such as e.g. cottonseed oil, sesame oil,coconut oil or peanut oil. Suitable dispersing or suspending agents foraqueous suspensions include synthetic or natural gums such astragacanth, alginate, gum arabic, dextran, sodiumcarboxymethylcellulose, gelatin, methylcellulose andpolyvinylpyrrolidone. The active ingredient may also be administered inthe form of a bolus, electuary or paste.

In addition to the compositions described above, the compositions of theinvention may also be formulated as a depot preparation. Suchlong-acting compositions may be administered by implantation (e.g.subcutaneously, intraabdominally, or intramuscularly) or byintramuscular injection. Thus, for example, the active ingredient may beformulated with suitable polymeric or hydrophobic materials (forexample, as an emulsion in a pharmaceutically acceptable oil), or an ionexchange resin.

The compounds of this invention either alone or in combination with eachother or other compounds generally will be administered in a convenientcomposition. The following representative composition examples areillustrative only and are not intended to limit the scope of the presentinvention. In the compositions that follow, “active ingredient” means acompound of this invention.

As used herein, “therapeutically effective time window” means the timeinterval wherein administration of the compounds of the invention to thesubject in need thereof reduces or eliminates the deleterious effects orsymptoms. In a preferred embodiment, the compound of the invention isadministered proximate to the deleterious effects or symptoms.

Nigella sativa Linn. (family: Ranunculacease), commonly known as blackseed or black curcumin, is an annual plant that has been traditionallyused in the Indian subcontinent, Arabian countries, and Europe forculinary and medicinal purposes as a natural remedy for a number ofillnesses and conditions that include asthma, hypertension, diabetes,inflammation, cough, bronchitis, headache, eczema, fever, dizziness andinfluenza. The seeds or its oil are used as a carminative, diuretic,lactoagogue, and vermifuge. They are also used in food as a spice and acondiment.

N. sativa seeds contain 36-38% fixed oils, proteins, alkaloids, saponinand 0.4-2.5% essential oil. The fixed oil is composed mainly ofunsaturated fatty acids, including the unusual C20:2 arachidic andeiosadienoic acids. Major components of the essential oil includethymoquinone (27-57%), p-cymene (7.1-15.5%), carvacrol (5.8-11.6%),trans-anethole (0.25-2.3%) p-terpineol (2.0-6.6%) and longifoline(1.0-8.0%). TQ readily dimerizes to form dithymoquinone and as usedherein, TQ will also refer to the naturally occurring dimmerdithymoquinone.

Many studies have been conducted, particularly during the past twodecades, on the effect of N. sativa seed extracts on various bodysystems in vivo or in vitro. Included among those physiologicalvariables studied are antioxidant, anti-inflammatory and analgesicactions, anticarcinogenic activity, hypotensive, antidiabetic,antiulcer, antimicrobial and antiparasitic responses [Ali B H, BlundenG. 2003. Pharmacological and toxicological properties of Nigella sativa.Phytother Res 17: 299-305]. This body of research teaches thatextraction methodology is a primary determinant of the effectiveness ofthe resulting N. sativa seed extract [see for example: Kokoska, L., J.Havlik, et al. (2008). “Comparison of chemical composition andantibacterial activity of Nigella sativa seed essential oils obtained bydifferent extraction methods.” J Food Prot 71(12): 2475-2480].

Recently studies have been reported using supercritical liquid extractsof N. sativa seeds containing 2.0 to 2.8 percent TQ, but none withextracts produced under the conditions of pressure, temperature and timeas described herein. Further, biological activities of extracts in theseand other studies with organic solvent extraction were roughly equal totheir TQ content and none of the reported studies were applicable tocommercially-scaled quantities of N. sativa extract [Ismail, M., G.Al-Naqeep, et al. (2010). “Nigella sativa thymoquinone-rich fractiongreatly improves plasma antioxidant capacity and expression ofantioxidant genes in hypercholesterolemic rats.” Free Radic Biol Med48(5): 664-672; Al-Naqeep, G., M. Ismail, et al. (2009). “Regulation ofLow-Density Lipoprotein Receptor and 3-Hydroxy-3-Methylglutaryl CoenzymeA Reductase Gene Expression by Thymoquinone-Rich Fraction andThymoquinone in HepG2 Cells.” J Nutrigenet Nutrigenomics 2(4-5):163-172].

As used herein, “commercial-scale quantities” of N. sativa seed areconsidered quantities of raw material in excess of about 10 kg. Allsupercritical fluid extraction herein refers to extraction ofcommercial-scale quantities of N. sativa powdered seed.

All prior art, including reports using supercritical extraction of about100 g or less of N. sativa, teach that TQ is the active component of N.sativa. The present application, however, teaches that certainsupercritical CO₂ extracts produced at pressures below 600 bar extractTQ more efficiently and exhibit unexpected potency greater than their TQcontent. Additionally, the present application teaches that whencommercial volumes of N. sativa seeds are extracted, supercritical CO₂extracts can be enriched with TQ using lower pressures withoutsubsequent extraction steps.

The present compositions can be provided in any convenient form. It canbe provided as dietary supplement in capsule or tablet form. It can beformulated into a food or drink, and provided, for example, as a snackbar, a cereal, a drink, a gum, or in any other easily ingested form. Itcan also be provided as a cream or lotion for topical application. Onetrained in the art can readily formulate the present composition intoany of these convenient forms for oral or topical administration.

The amount of other additives per unit serving are a matter of designand will depend upon the total number of unit servings of thenutritional supplement daily administered to the patient. The totalamount of other ingredients will also depend, in part, upon thecondition of the patient. Preferably, the amount of other ingredientswill be a fraction or multiplier of the RDA or DRI amounts. For example,the nutritional supplement will comprise 50% RDI (Reference DailyIntake) of vitamins and minerals per unit dosage and the patient willconsume two units per day.

Flavors, coloring agents, spices, nuts and the like can be incorporatedinto the product. Flavorings can be in the form of flavored extracts,volatile oils, chocolate flavorings (e.g., non-caffeinated cocoa orchocolate, chocolate substitutes such as carob), peanut butterflavoring, cookie crumbs, crisp rice, vanilla or any commerciallyavailable flavoring. Flavorings can be protected with mixed tocopherols.Examples of useful flavorings include but are not limited to pure aniseextract, imitation banana extract, imitation cherry extract, chocolateextract, pure lemon extract, pure orange extract, pure peppermintextract, imitation pineapple extract, imitation rum extract, imitationstrawberry extract, or pure vanilla extract; or volatile oils, such asbalm oil, bay oil, bergamot oil, cedarwood oil, cherry oil, walnut oil,cinnamon oil, clove oil, or peppermint oil; peanut butter, chocolateflavoring, vanilla cookie crumb, butterscotch or toffee. In a preferredembodiment, the nutritional supplement contains berry or other fruitflavor. The food compositions may further be coated, for example with ayogurt coating if it is as a bar.

Emulsifiers may be added for stability of the final product. Examples ofsuitable emulsifiers include, but are not limited to, lecithin (e.g.,from egg or soy), or mono- and di-glycerides. Other emulsifiers arereadily apparent to the skilled artisan and selection of suitableemulsifier(s) will depend, in part, upon the formulation and finalproduct.

Preservatives may also be added to the nutritional supplement to extendproduct shelf life. Preferably, preservatives such as potassium sorbate,sodium sorbate, potassium benzoate, sodium benzoate or calcium disodiumEDTA are used.

In addition to the carbohydrates described above, the nutritionalsupplement can contain natural or artificial sweeteners, e.g., glucose,sucrose, fructose, saccharides, cyclamates, aspartamine, sucralose,aspartame, acesulfame K, or sorbitol.

Manufacture of the Preferred Embodiments

The nutritional supplements of the present invention may be formulatedusing any pharmaceutically acceptable forms of the vitamins, mineralsand other nutrients discussed above, including their salts. They may beformulated into capsules, tablets, powders, suspensions, gels or liquidsoptionally comprising a physiologically acceptable carrier, such as butnot limited to water, milk, juice, soda, starch, vegetable oils, saltsolutions, hydroxymethyl cellulose, carbohydrate. In a preferredembodiment, the nutritional supplements may be formulated as powders,for example, for mixing with consumable liquids, such as milk, juice,sodas, water or consumable gels or syrups for mixing into othernutritional liquids or foods. The nutritional supplements of thisinvention may be formulated with other foods or liquids to providepre-measured supplemental foods, such as single serving beverages orbars, for example.

In a particularly preferred embodiment, the nutritional supplement willbe formulated into a nutritional beverage, a form that has consumerappeal, is easy to administer and incorporate into one's daily regimen,thus increasing the chances of patient compliance. To manufacture thebeverage, the ingredients are dried and made readily soluble in water.For the manufacture of other foods or beverages, the ingredientscomprising the nutritional supplement of this invention can be added totraditional formulations or they can be used to replace traditionalingredients. Those skilled in food formulating will be able to designappropriate foods or beverages with the objective of this invention inmind.

The nutritional supplement can be made in a variety of forms, such aspuddings, confections, (i.e., candy), nutritional beverages, ice cream,frozen confections and novelties, or non-baked, extruded food productssuch as bars. The preferred form is a powder to add to a beverage or anon-baked extruded nutritional bar. In another embodiment, theingredients can be separately assembled. For example, certain of theingredients (e.g., the conjugated fatty acids or alcohols and thiolcompounds) can be assembled into a tablet or capsule using knowntechniques for their manufacture. The remaining ingredients can beassembled into a powder or nutritional bar. For the manufacture of afood bar, the dry ingredients are added with the liquid ingredients in amixer and mixed until the dough phase is reached; the dough is put intoan extruder and extruded; the extruded dough is cut into appropriatelengths; and the product is cooled. The two assembled forms comprise thenutritional supplement and can be packaged together or separately, suchas in the form of a kit, as described below. Further, they can beadministered together or separately, as desired.

Use of Preferred Embodiments

The preferred embodiments contemplate treatment of disorders related tooxidative stress, inflammation, metabolic syndrome, all forms ofdiabetes, and obesity. A pharmaceutically acceptable carrier may also beused in the present compositions and formulations. The recommended dailyamounts of each ingredient, as described above, serve as a guideline forformulating the nutritional supplements of this invention. The actualamount of each ingredient per unit dosage will depend upon the number ofunits administered daily to the individual in need thereof. This is amatter of product design and is well within the skill of the nutritionalsupplement formulator.

The ingredients can be administered in a single formulation or they canbe separately administered. For example, it may be desirable toadminister the compounds in a form that masks their taste (e.g., capsuleor pill form) rather than incorporating them into the nutritionalcomposition itself (e.g., powder or bar). Thus, the invention alsoprovides a pharmaceutical pack or kit comprising one or more containersfilled with one or more of the ingredients of the nutritionalcompositions of the invention (e.g., nutritional supplement in the formof a powder and capsules containing TQ and/or synephrine). Optionallyassociated with such container(s) can be a notice in the form prescribedby a government agency regulating the manufacture, use or sale ofpharmaceutical products, which notice reflects approval by the agency ofmanufacture, use of sale for human administration. The pack or kit canbe labeled with information regarding mode of administration, sequenceof administration (e.g., separately, sequentially or concurrently), orthe like. The pack or kit may also include means for reminding thepatient to take the therapy. The pack or kit can be a single unit dosageof the combination therapy or it can be a plurality of unit dosages. Inparticular, the agents can be separated, mixed together in anycombination, present in a formulation or tablet.

The preferred embodiments provide compositions and methods to promotenormal adipocyte or myocyte function. All preferred embodiments providevarying amounts of N. sativa essential oil containing from about 0.01 to39% (w/w) TQ as a marker compound. Generally the formulations comprisesupercritical CO₂ extracts of ground N. sativa seeds. In one embodiment,the composition comprises a supercritical CO₂-extracted fraction ofground N. sativa seeds initially extracted at 140 bar, 50° C. and 30minutes.

A second embodiment comprises a supercritical CO₂-extracted fraction ofground N. sativa seeds extracted at 140 bar, 50° C. and 120 minutesfollowing the removal of the first fraction. A third embodimentcomprises a supercritical CO₂-extracted fraction of ground N. sativaseeds extracted at 300 bar, 60° C. and 180 minutes following the removalof the second fraction.

Finally, a fourth embodiment comprises the combination resulting fromthe addition of the third fraction to the spent, extracted ground seedin a ratio of 1:24 (Extract:Spent Seed Powder).

EXAMPLES Example 1 Efficiency of Supercritical Fluid Extraction ofNigella sativa from Research-Scale (100 g) and Commercial-Scale (100 kg)Quantities of Powdered Seeds

Objective—

The first objective of this experiment was to compare the extractionefficiency of supercritical CO₂ extraction of powdered N. sativa seedsunder research conditions, defined as amounts of about 100 g, with theextraction efficiency under commercial-scale conditions, defined asamounts of about 100 kg. A second objective was to produce foursupercritical CO₂ extracts of N. sativa seeds obtained under variousconditions of pressure, temperature and time from commercial-scalequantities. To date, no supercritical extraction procedure hasdemonstrated the ability to process commercial quantities (1-100 kg) ofpowdered seed that contain concentrations of TQ above 10 percent withoutfurther extraction steps. It is well known in the art that scale of theextraction process can greatly affect the components of the extract andthat extraction results obtained with small quantities of substrate donot reflect results obtained with larger, commercial quantities.

As TQ is widely considered the putative active compound in N. sativaessential oil, the TQ content of each of the fractions was used forcomparing the efficiency of the supercritical extraction process underresearch and commercial conditions. Additionally, the use of the TQstandard allows for comparison to prior art.

Raw Material Purchase—

The black seeds of N. sativa are seasonal, available from April to Juneof every year. The main source is in northern parts of Indiaparticularly in Uttar Pradesh. Stocked material is available through outthe year. The cost generally fluctuates between $2.5/kg to $ 5.0/kg.

Grinding and Sieving—

Once the 110 kg of N. sativa seeds was purchased it was ground to a finepowder between 20-30 mesh. A magnetic screening system was used toensure removal of metallic impurities, particularly iron.

Supercritical Fluid Extraction—

In this example, the process conditions during the extraction of N.sativa seeds with supercritical CO₂ extraction were varied with respectto pressure, temperature and time (Table 2). The commercial-scaleextraction was performed in a polyvalent pilot plant extraction set-upshown schematically in FIG. 3. Liquid CO₂ entering the apparatus wascooled in condenser C before it was pressurized and passed into thesystem. The flow rate was adjusted manually before the experiment.During the extraction process, the temperatures of the extractor, CO₂,and separators 1 and 2 (S1/S2) were automatically regulated through therecirculation of thermostatic water from three individually regulatedwater baths. Supercritical CO₂ extraction under research conditions wasperformed using a laboratory-scale SFE apparatus (100 mL up to 10 Lsystems). Extraction conditions and processes were similar for bothcommercial and research scaling as follows.

The ground N. sativa seeds were loaded into a cylindrical container thatwas equipped with steel mesh filters on both ends, thus enabling CO₂ topass the cylinder without transport of solids to the exterior. Afterprepressurization of the total system and the regulation of the CO₂ flowrate, the extractor (EXT) was depressurized and the cylinder wassubsequently placed inside the extractor, after which the complete CO₂flow was redirected toward the extractor using valves V1 and V2. Thetemperature/pressure combinations of both separator vessels S1 and S2were controlled individually. The extraction was stopped by redirectingthe CO₂ flow again to recirculation over the condenser. The solidresidue was removed from the extractor after stepwise depressurizationof the entire system. Subsequently, both separator vessels were rinsedwith hexane, and extracts were collected in UV-opaque bottles to preventUV-activated degradation of the extract. Pressure/temperaturecombinations and extraction times for the extracts produced (NS8-NS10)are presented in Table 2.

Thymoquinone Concentration—

TQ content of the various fractions obtained were determined by HPLCanalysis as described by Ghosheh (Ghosheh O A, Houdi A A, Crooks P A.High performance liquid chromatographic analysis of thepharmacologically active quinones and related compounds in the oil ofthe black seed (Nigella sativa L.). J Pharm Biomed Anal. April 1999;19(5):757-762) with no modifications.

Results—

Seven fractions 1, 2, 3, 4, 5, 6 and 7 were initially collected duringthe three-hour extraction process. Fractions 1, 2, 3 and 4, which werecollected over 30 minutes at 140 bar and 50° C. in Sep 1, were combinedto produce the fraction termed NS8 with a TQ content, respectively, of5.47% and 2.24% for research and commercial-scale extraction. The fifthworking fraction, which was collected over the next two-hours at 140 barand 50° C. in Sep 2, was termed NS9 and contained, respectively, 2.95%and 39.3% TQ % for research and commercial-scale extraction.

Following extraction at 140 bar and 50° C., the pressure was increasedto 300 bar and temperature to 60° C. over three hours producingfractions 6 and 7 that were combined to form fraction NS10 with a TQcontent for research and commercial scale of 1.29% and 0.24%,respectively (Table 2).

Finally, a fourth, novel TQ-containing composition Spent#2 was createdby combining fractions NS10 with the extracted (Spent#1) N. sativapowdered seed in a ratio of 24:1 (extracted seed powder:NS10). Theresulting TQ concentration of this sample was 0.01%. The extractionprotocol is represented schematically in FIG. 4.

Relative to the research-scale process and prior art, thecommercial-scale, supercritical CO₂ extraction process demonstratedremarkable efficiency in extraction of TQ in the production of NS9.Unexpectedly, using a low-pressure, single-step extraction process, thecommercial-scale extraction produced a fraction containing nearly 40% TQ(NS9). This dramatic increase in TQ for extract NS9 was apparently dueto reduced TQ recovery from the earlier fraction NS8 and resulted inlower TQ in the subsequent NS10 fraction.

TABLE 2 Contrasting Thymoquinone Content of Supercritical Carbon DioxideExtraction of Research Quantities (100 g) and Commercial-ScaleQuantities (100 kg) of N. sativa Seed Powder Under Various ExtractionConditions of Pressure, Temperature and Time Thymoquinone Processconditions Content (% w/w) Pressure Temperature Time 100 g 100 kg Sample(Bar) (° C.) (min) Extracted Extracted NS8 (1/2/3/4) 140 50 30 5.47 2.24NS9 (5) 140 60 120 2.95 39.3 NS10 (6/7) 300 60 180 1.29 0.24

As commercial-scaled supercritical CO₂ extraction of N. sativa has neverbefore been attempted, these fractions were considered novel and placedinto a screening program to assess potential health benefits. Results ofthe screening of these novel extracts are presented in the followingExamples.

Example 2 Free Radical Scavenging Activity of Supercritical CarbonDioxide Extract NS9 of Nigella sativa is Greater than Thymoquinone

Objective—

The objective of this experiment was to compare the antioxidant (freeradical scavenging) activity of the four NS fractions obtained bysupercritical CO₂ extraction of N. sativa powdered seeds in Example 1with the pure TQ marker compound.

Chemicals—

TQ, 2,2-diphenyl-1-picrylhydrazyl and all other compounds used in thisexample were purchased from Sigma (St. Louis, Mo.) and were of thehighest purity commercially available. The four N. sativa samples usedin this study were those commercial-scale extracts described in Example1.

Methodology—

Antioxidant activity was determined utilizing2,2-diphenyl-1-picrylhydrazyl (DPPH), which is a stable radical. The oddelectron in the DPPH free radical gives a strong absorption maximum at550 nm and is purple in color. The color turns from purple to yellow asthe molar absorption of the DPPH radical at 550 nm is reduced when theodd electron of DPPH radical becomes paired with hydrogen from a freeradical scavenging antioxidant to form the reduced DPPH-H. The testsamples were dissolved in methanol containing 1% dimethyl sulfoxide andadded to microtiter wells in 100 μL aliquots to 100 μL of a 100 μM DPPHsolution in methanol. Readings were taken at 10, 30 and 60 minutesfollowing the addition of the test material. Percent inhibition of theDPPH radical by the test material was computed relative to theinhibition of the DPPH radical by the vitamin E analog trolox andtabulated as μmol trolox/g test material.

Results—

Only NS9 of the four TQ-containing supercritical extracts exhibitedantioxidant activity in the DPPH assay (Table 3). The antioxidantactivity exhibited by NS9, however, was 3.3-times the activity seen withpure TQ. When compared to the literature, this result underscores thedifference between supercritical CO₂ essential oil extracts of N. sativaand those resulting from use of organic solvents or steam distillation.For example, the DPPH-reducing activity of the essential oil fraction ofN. sativa, produced by light petroleum Soxhlet extraction followed bysteam distillation and containing 48% TQ, was reported to be less thanone-half that of the TQ standard. When adjusted for TQ content, however,it appeared that all of the antioxidant activity of the essential oilfraction was due to TQ. (Burits, M.; Bucar, F., Antioxidant activity ofNigella sativa essential oil. Phytother Res 2000, 14 (5), 323-8). Thus,although the TQ content of the cited published study was 48% compared to39% for fraction NS9, the antioxidant activity of the supercritical CO₂N. sativa extract NS9 was quantitatively superior to the both theorganic extract and TQ. This example demonstrates that the chemicalbehavior of N. sativa seed extracts is largely a function of extractionconditions such as solvent, temperature and time and independent of TQconcentration.

TABLE 3 Antioxidant Activity of Supercritical Carbon Dioxide Fractionsof N. sativa Seeds Relative to Thymoquinone and Petroleum ExtractedFraction of N. sativa Seeds Thymoquinone DPPH Reducing Activity(1) TestMaterial Content [%] [μmol Trolox/g TQ] Thymoquinone 100 45.2 (Sigma)Essential Oil 48 43.2 (Petroleum Ext)² NS8 2.24 No activity up to 1000μg/mL NS9 39.3 148 NS10 0.24 No activity up to 1000 μg/mL Spent#1 0.00No activity up to 1000 μg/mL NSSpent#2 0.010 No activity up to 1000μg/mL ( ))Computed on basis of TQ content. ²Burits, M.; Bucar, F.,Antioxidant activity of Nigella sativa essential oil. Phytother Res2000, 14 (5), 323-8.

Conclusion—

A supercritical CO₂ essential oil extract of N. sativa collected at 140bar, 60° C. and 120 minutes and containing about 39% TQ possessed overthree times the free radical savaging activity of TQ alone. As the priorart teaches that the TQ content of the essential oil fraction of N.sativa solely contributes to the antioxidant activity, unexpectedly, theunique combination of compounds extracted from N. sativa in NS 9 behavessynergistically to greatly exceed the antioxidant activity of theputative active component TQ.

Example 3 Inhibition of Prostate Specific Antigen Secretion from LNCaPProstate Cells by Supercritical Carbon Dioxide Extracts of Nigellasativa Differs Qualitatively and Quantitatively from Thymoquinone

Objective—

While the effects of TQ and various extracts of N. sativa have showncytotoxicity to a number of tumor cell lines, no studies have been doneon the effect of TQ-containing extracts of N. sativa on the secretion ofprostate specific antigen (PSA), a marker for prostate hyperplasia aswell as cancer. While PSA is present in small quantities in the serum ofnormal men, it is often elevated in the presence of nonproliverative aswell as neoplastic prostate disorders. Examples of noncancerous ornonproliverative prostate disorders exhibiting elevated PSA are benignprostate hyperplasia and infections of the prostate. The objective ofthis experiment was to determine whether commercial-scale, supercriticalfluid CO₂ extracts of N. sativa containing TQ would affect prostatespecific antigen secretion from non-proliferating prostate cells in amanner similar to TQ alone.

Chemicals—

PSA was quantified from the cell culture supernatant fluid using theQuantikine Human KLK3/PSA Immunoassay kit (R&D Systems, Inc.,Minneapolis, Minn.). All other materials used in this example werepurchased from Sigma (St. Louis, Mo.) or otherwise noted and were of thehighest purity commercially available. The N. sativa samples used inthis study were those commercial-scale extracts described in Example 1.

Cell Culture and Treatment—

The LNCaP prostate cell line, which produces PSA, was used to study theeffects of commercial-scale N. sativa scale extracts of Example 1 andpure TQ on the secretion of PSA. LNCaP cells were purchased from theAmerican Type Culture Collection (ATCC, Manassas, Va.) and sub-culturedaccording to instructions supplied by ATCC. Prior to experiments, cellswere cultured in DMEM containing 10% FBS-HI added 50 units penicillin/mland 50 μg streptomycin/ml, and maintained in log phase prior toexperimental setup. Cells were grown in a 5% CO₂ humidified incubator at37° C. Components of medium included: (1) 10% FBS/DMEM (Fetal BovineSerum/Dulbecco's Modified Eagle's Medium) containing 4.5 g glucose/L;(2) 50 U/ml penicillin; and (3) 50 μg/ml streptomycin. Growth medium wasmade by adding 50 ml of heat inactivated FBS and 5 ml ofpenicillin/streptomycin to 500 ml DMEM. This medium was stored at 4° C.Before use, the medium was warmed to 37° C. in a water bath.

LNCaP cells were seeded at an initial density of 6×10⁴ cells/cm² in96-well plates. For two days, the cells were allowed grow to reachconfluence. On day three post seeding, cells were treated with TQ, NS8,NS9, NS10, Spent#1, and NSSpent#2 at the concentrations listed in FIGS.5, 6 and 7. Twenty-four hours later, the supernatant media were sampledand assayed for PSA.

TABLE 4 Summary of Effects on Prostate Specific Antigen Secretion byLNCaP Prostate Cells by Supercritical Extracts of Nigella sativa Seedsand Pure Thymoquinone Test Material Effect on PSA Secretion over 24Hours Thymoquinone Decreased PSA at 5 and 10 μg/mL, dramatic (Sigma)increase in PSA secretion at 50 and 100 μg/mL. NS8 Increased PSAsecretion at all doses, but decreased with increasing NS8. NS9Dose-related decrease in PSA from 5 to 100 μg/mL. NS10 Dose-relatedincrease in PSA from 5 to 100 μg/mL. Spent#1 Increase in PSA secretionat both doses tested 250 and 500 μg/mL. NSSpent#2 Decrease in PSAsecretion at both doses tested 250 and 500 μg/mL.

Results—

FIGS. 5, 6 and 7 depict the pair-wise contrasts, respectively, of TQ vsNS9, NS8 vs NS10, and Spent#1 vs NSSpent#2. Unexpectedly, TQ did notexhibit a linear dose-response curve and decreased PSA only at the twolowest doses tested while dramatically increasing PSA secretion at thehighest doses, 166 and 207%. The response of NS9 differed qualitativelyfrom that of TQ alone. NS9 produced a linear dose-related decrease inPSA over all four concentrations (FIG. 5). NS8 and NS 10 differed intheir effect on PSA secretion from both TQ and NS9. NS8 increased PSA atall doses, but this effect decreased with increasing NS8 concentration(FIG. 6). Conversely, NS10 increased PSA at all doses in a directdose-response relationship. Finally, while Spent#1 increased PSA at both250 and 500 mg/mL, NSSpent#2 nearly totally decreased PSA secretion atboth of these concentrations.

A summary of the various effect of N. sativa supercritical CO₂ extractson PSA secretion is presented in Table 4. It is clear from this summarythat these extracts produce novel effects on PSA secretion from prostatecells implying differences in composition and biological activity notanticipated in the prior art.

Example 4 Select Commercial Supercritical Fluid Extracts of Nigellasativa are Potent Uncouplers of Mitochondrial Membrane Potential in3T3-L1 Adipocytes

Objective—

The objective of this experiment was to determine whethercommercial-scale, supercritical CO₂ extracts of N. sativa powdered seedsproduced in Example 1 directly reduce mitochondria membrane potential in3T3-L1 adipocytes compared to pure TQ or DNP.

The Model—

3T3-L1 murine fibroblast are routinely used to study the potentialeffects of compounds on white adipose tissue in vitro. This cell lineallows investigation of stimuli and mechanisms that regulateinflammatory mediators of cytokine secretion of the adipocyte. Aspreadipocytes, 3T3-L1 cells have a fibroblastic appearance. Theyreplicate in culture until they form a confluent monolayer, after whichcell-cell contact triggers G_(o)/G₁ growth arrest. Terminaldifferentiation of 3T3-L1 cells to adipocytes depends on proliferationof both pre- and post-confluent preadipocytes. Subsequent stimulationwith 3-isobutyl-1-methylxanthane, dexamethasone, and high does ofinsulin (MDI) for two days prompts these cells to undergo post-confluentmitotic clonal expansion, exit the cell cycle, and begin to expressadipocyte-specific genes. Approximately five days after induction ofdifferentiation, more than 90% of the cells display the characteristiclipid-filled adipocyte phenotype. At this stage of differentiation,response to mitochondrial uncouplers such as DNP may be assessed.

Chemicals—

2,4-Dinitrophenol and all other chemicals used in this example werepurchased from Sigma (St. Louis, Mo.) or otherwise noted and were of thehighest purity commercially available. The N. sativa commercial-scaleextracts used in this study were those described in Example 1.

Cell Culture and Treatment—

The murine fibroblast cell line 3T3-L1 was purchased from the AmericanType Culture Collection (Manassas, Va.) and sub-cultured according toinstructions from the supplier. Prior to experiments, cells werecultured in DMEM containing 10% FBS-HI added 50 units penicillin/ml and50 μg streptomycin/ml, and maintained in log phase prior to experimentalsetup. Cells were grown in a 5% CO₂ humidified incubator at 37° C.Components of the pre-confluent medium included: (1) 10% FBS/DMEM (FetalBovine Serum/Dulbecco's Modified Eagle's Medium) containing 4.5 gglucose/L; (2) 50 U/ml penicillin; and (3) 50 μg/ml streptomycin. Growthmedium was made by adding 50 ml of heat inactivated FBS and 5 ml ofpenicillin/streptomycin to 500 ml DMEM. This medium was stored at 4° C.Before use, the medium was warmed to 37° C. in a water bath.

3T3-T1 cells were seeded at an initial density of 6×10⁴ cells/cm² in96-well plates. For two days, the cells were allowed grow to reachconfluence. Following confluence, the cells were forced to differentiateinto adipocytes by the addition of differentiation medium; this mediumconsisted of (1) 10% FBS/DMEM (high glucose); (2) 0.5 mMmethylisobutylxanthine; (3) 0.5 μM dexamethasone and (4) 10 μg/mlinsulin (MDI medium). After three days, the medium was changed topost-differentiation medium consisting of 10 μg/ml insulin in 10%FBS/DMEM.

Treatment with 2,4-Dinitrophenol and Test Material—

On Day 6 post differentiation, DNP or test materials TQ, NS8, NS9, orNS10 were dissolved in DMSO and added to the culture medium to achieveconcentrations of 500 μM for DNP and 25 μg/mL for the test materialseach in eight wells of a single column 60 min at 37° C. JC-1 was thenadded to the test and negative control columns in 10 μL DMSO to achievea final concentration of 5 μM and allowed to incubate at 37° C. for anadditional 30 min. A DMSO and solvent plus JC-1 control were runconcurrently with each experiment. A Packard Fluorocountspectrofluorometer (Model#BF10000, Meridan, Conn.) set at 560 nmexcitation and 590 nm emission was used for quantification of aggregatefluorescence and at 485 nm excitation/530 emission for monomerfluorescence.

Measuring Mitochondrial Membrane Potential Changes (ΔΨm)—

JC-1 (Sigma, St. Louis, Mo.) has advantages over other cationic dyes inthat it can selectively enter into mitochondria and reversibly changecolor from green to red as the membrane potential increases. In healthycells with high mitochondrial membrane potential (ΔΨm), JC-1spontaneously forms complexes known as J-aggregates with intense redfluorescence. On the other hand, in cells with low ΔΨm, JC-1 remains inthe monomeric form exhibiting only green fluorescence. The changes inΔΨm by different forms of JC-1 as either green or red fluorescence areboth quantified by a fluorescence plate reader with appropriate filtersets.

Calculation of Relative Decrease in Mitochondrial Membrane Potential—

Aggregate and monomer fluorescence was computed for the 500 μM DNPpositive control as well as the test materials TQS, NS8, NS9 or NS10relative to JC-1 negative controls. The ratio of the monomer toaggregate relative fluorescence was then determined as a measure ofrelative decrease in ΔΨm. For statistical comparisons, 95% confidenceintervals were computed (Excel, Microsoft, Redman, Wash.) and graphed(FIG. 8) with the mean relative monomer/aggregate ratios. By utilizingthe 95% confidence intervals, the probability of a type I error was setat the nominal 5% level.

Results—

The positive control DNP at 500 μM decreased in ΔΨm in 3T3-L1 adipocytesto a similar extent as 25 μg/mL pure TQ, approximately 30 to 40% (FIG.8). The N. sativa commercial-scale extracts NS8, NS9 and NS10, however,decreased mitochondrial membrane potential 3.7- to 4.1-fold relative tosolvent controls. This example further demonstrates that thecommercial-scale, supercritical CO₂ extracts of N. sativa possessbiological activity unexplained by TQ content alone.

Example 5 Select Supercritical Fluid Extracts of Nigella sativa InhibitInflammation-Stimulated Nitric Oxide Biosynthesis in Adipocytes andMyocytes

Objective—

The objective of this experiment was to determine whether thecommercial-scale, supercritical CO₂ extracts of N. sativa seeds obtainedin Example 1 reduce inflammation-induced NO secretion in adipocytes andmyocytes as effectively as TQ alone.

The Model—

The murine 3T3-L1 preadipocyte and murine C2C12 premyocyte models wereused in this Example. This model was selected to serve as the surrogatefor adipocytes and myocytes that are exposed to a variety of theinflammatory stimuli of invading bacteria, modeled by lipopolysaccharide(LPS), as well as the counter-inflammatory responses of infiltratingmacrophage, modeled by interferon gamma (IFγ) and tumor necrosis factoralpha (TNFα). Additionally alterations in NO levels have beendemonstrated in pathologic conditions in humans such as obesity,diabetes, hypertension, osteoarthritis, osteoporosis, and interstitialcystitis.

Chemicals—

Heat-inactivated fetal bovine serum (HIFBS), penicillin and streptomycinsolution, and Dulbecco's Modification of Eagle's Medium (DMEM) werepurchased from Mediatech (Herndon, Va.).2-N-7-(nitrobenz-2-oxa-1,3-diazol-4-yl)amino-2-deoxy-d-glucose (2-NBDG)and N-methyl-4-hydrazino-7-nitrobenzofurazan (NBDM) were obtained fromInvitrogen (Carlsbad, Calif.). TQ, bacterial lipopolysaccharide (LPS),murine TNFα and Interferon-γ and all standard chemicals, unless noted,were obtained from Sigma (St Louis, Mo.) and were of the highest puritycommercially available.

Cell Culture and Treatment—

The murine fibroblast cell line 3T3-L1 was purchased from the AmericanType Culture Collection (Manassas, Va.) and sub-cultured according toinstructions from the supplier. Prior to experiments, cells werecultured in DMEM containing 10% FBS-HI added 50 units penicillin/ml and50 μg streptomycin/ml, and maintained in log phase prior to experimentalsetup. Cells were grown in a 5% CO₂ humidified incubator at 37° C.Components of the pre-confluent medium included: (1) 10% FBS/DMEM (FetalBovine Serum/Dulbecco's Modified Eagle's Medium) containing 4.5 gglucose/L; (2) 50 U/ml penicillin; and (3) 50 μg/ml streptomycin. Growthmedium was made by adding 50 ml of heat inactivated FBS and 5 ml ofpenicillin/streptomycin to 500 ml DMEM. This medium was stored at 4° C.Before use, the medium was warmed to 37° C. in a water bath.

3T3-T1 cells were seeded at an initial density of 6×10⁴ cells/cm² in24-well plates. For two days, the cells were allowed grow to reachconfluence. Following confluence, the cells were forced to differentiateinto adipocytes by the addition of differentiation medium; this mediumconsisted of (1) 10% FBS/DMEM (high glucose); (2) 0.5 mMmethylisobutylxanthine; (3) 0.5 μM dexamethasone and (4) 10 μg/mlinsulin (MDI medium). After three days, the medium was changed topost-differentiation medium consisting of 10 μg/ml insulin in 10%FBS/DMEM.

For assessing the effects of the N. sativa extracts oncytokine-stimulated NO-production in 3T3-L1 adipoctyes, D6 adipoctyeswere treated with 50, 10, 5, or 1 μg TQ or N. sativa extract/mL for 1hour and then stimulated with a cytokine mixture containing 1 μg LPS/mL,50 ng TNFα/mL and 100 U IFγ/mL (LTI) for 20 hours. L-N^(G)-Nitroargininemethyl ester (L-NAME) at 200 μM (47 μg/mL) was used as the positivecontrol and 0.1% DMSO for the negative control.

Mouse C2C12 myoblasts (American Type Culture Collection, Manassas, Va.)were maintained in Dulbecco's modified Eagle's medium (DMEM)supplemented with 10% fetal calf serum (FCS), 50 units/ml penicillin and50 μg/ml streptomycin. As cells reached confluence, the medium wasswitched to the differentiation medium containing DMEM and 2% horseserum. Medium was changed every other day. After 4 additional days, thedifferentiated C2C12 cells had fused into myotubes, which were thentreated in serum-free DMEM with either vehicle alone (0.1% DMSO), or 50,10, 5, or 1 μg TQ or N. sativa extract/mL for 1 hour and then stimulatedwith a cytokine mixture containing 1 μg LPS/mL, 50 ng TNFα/mL and 100 UIFγ/mL (LTI) for 20 hours. At the end of the treatment period, mediawere sampled for determination of NO.

Nitric Oxide Determination—

NBD methylhydrazine (NBDM, N-methyl-4-hydrazino-7-nitrobenzofurazan) wasused to detect N-methyl-4-amino-7-nitrobenzofuazan, the fluorescentproduct of the NBDM reagent with nitrite (Buldt A, Karst U.Determination of nitrite in waters by microplate fluorescencespectroscopy and HPLC with fluorescence detection. Anal Chem. Aug. 11999; 71(15):3003-3007). In a separate, black-walled, 96-well,microtiter plate, a 7 μL aliquot of a 4.8×1 mol NBDM/L solution wasadded to 200 μL of the supernatant media followed by the addition of 15μL of concentrated phosphoric acid. After a reaction time of 30 minutesat ambient temperature, the fluorescence was read with 485 nm excitationfilter and a 530 nm emission filter in a Packard Fluorocountspectrofluorometer (Model#BF10000, Meridan, Conn.). Fluoresence waslinear in the range of 3.59×10⁻⁷ to 1.44×10⁻⁵ mol nitrite/L. Thestandard deviation was 3.5 percent for 1.44×10⁻⁶ mol nitrite/L.Experiments were performed a minimum of three times with eightreplicates per dose, capturing the median inhibitory concentration(IC₅₀) when possible.

Calculations—

The median inhibitory concentrations (IC₅₀) and 95% confidence intervalwere calculated using CalcuSyn (BIOSOFT, Ferguson, Mo.). Thisstatistical package performs multiple drug dose-effect calculationsusing the Median Effect methods described by Chou and Talalay [Chou T C,Talalay P. Quantitative analysis of dose-effect relationships: thecombined effects of multiple drugs or enzyme inhibitors. Adv EnzymeRegul. 1984; 22:27-55].

TABLE 5 Median Inhibitory Concentrations of Three Supercritical FluidExtracts of N. sativa and Thymoquinone for iNOS- mediated NOBiosynthesis in Adipocytes and Myocytes 3T3-L Adipocytes* C2C12Myocytes* Test Sample [μg/mL] [μg/mL] TQS (100% TQ) 1.9 (1.4-2.7) 1.9(1.0-3.4) NS8 (2.24% TQ) 1.2† 0.11† NS9 (39.3% TQ) 1.6 1.6 NS10 (0.24%TQ) 6.5† 0.007† NSSpent#2 0.92† Not Tested *Value computed on the basisof TQ content. †Significantly different (p < 0.05) from TQS.

Results—

TQ alone was a potent inhibitor of iNOS-mediated NO production in bothadipocytes and myocytes stimulated with the trivalent cytokine mixtureand exhibited a median inhibitory concentrations of 1.9 μg TQ/mL in bothcell types (Table 5). These results are consistent with publishedresults for NO inhibition in the LPS-stimulated macrophages(IC₅₀=1.4-2.8 μg/mL). [El-Mahmoudy A, Matsuyama H, Borgan M A, et al. TQsuppresses expression of inducible nitric oxide synthase in ratmacrophages. Int Immunopharmacol. October 2002; 2(11):1603-1611]. Thefour supercritical extracts of N. sativa tested in this example NS8,NS9, NS10 and NSSpent#2 were all also potent inhibitors ofLTI-stimulated NO biosynthesis in 3T3-L1 adipocytes and C2C12 myocytes(NSSpent#2 not tested in myocytes). While NS8 and NS9 were equallypotent to TQ alone computed on the basis of TQ content, NSSpent#2 wasmore potent and NS10 less potent than TQ in adipocytes. Conversely, NS10was more potent than TQ in the C2C12 myocytes. Consistent with itsresponse in 3T3-L1 adipocytes NS8 was equally as potent as TQ in theC2C12 myocytes.

These unexpected results serve to demonstrate the superior NO inhibitorypotency of specific supercritical extracts of N. sativa over TQ alone.

Thus, the inhibitory effects of N. sativa supercritical fluid extractsin this example demonstrate a novel result encompassing multiplereceptor signaling pathways in adipocytes and myocytes. Such a findinghas thus far not been reported in the prior art.

Example 6 Select Supercritical Fluid Extracts of Nigella sativa IncreaseLypolysis in 3T3-L1 Adipocytes and C2C12 Myocytes

Objective—

The objective of this experiment was to determine whether thesupercritical fluid CO₂ extracts of N. sativa obtained in Example 1induce lipolysis in adipocytes or myocytes.

Chemicals—

All chemicals used in this example were purchased from Sigma (St. Louis,Mo.) or otherwise noted and were of the highest purity commerciallyavailable. The N. sativa samples used in this study were those describedin Example 1.

Cell Culture and Treatment—

Culture and treatment of 3T3-L1 adipocytes and C2C12 myocytes was asdescribed in Example 5. Concentrations of the forskolin positive controland test materials are listed in FIG. 9 for adipocytes and FIG. 10 formyocytes.

Glycerol Assay—

Free fatty acid release from 3T3-L1 adipocytes or C2C12 myocytes wasquantified by measuring glycerol secretion into the medium. Glycerol wasmeasured spectrophotometrically using the Free Glycerol DeterminationKit (F6428, Sigma) and an EL 312e Microplate BIO-KINETICSspectrophotometer (BioTek, Winooski, Vt.).

Data Analysis—

Glycerol release from adipocytes and myocytes was expressed,respectively, as the percent increase in free fatty acid secretion (FIG.9) and relative glycerol content (glycerol index FIG. 10) ±95%confidence intervals of eight observations for one of threerepresentative experiments.

Results—

The forskolin positive control, TQ and all NS supercritical extractsinduced free fatty acid release in adipocytes (FIG. 9) and myocytes(FIG. 10). Specifically, NS8 at 5 μg/mL induced similar free fatty acidsecretion from 3T3-L1 adipocytes as 82 μg/mL of the positive controlforskolin. NS9, exhibiting an increase in free fatty acid release of102% in adipocytes, was twice as potent as TQS (Sigma) on a weight basisand 4.8-times as potent based on a TQ content of 39%.

Example 7 Select Supercritical Fluid Extracts of Nigella sativa ActivateAMPK in Myocytes

Objective—

The objective of this experiment was to compare the effect of the AMPmimentic AICAR on AMPK activation in C2C12 myocytes with thecommercial-scale supercritical CO₂ extracts of N. sativa produced inExample 1.

The Model—

The C2C12 myocyte model as described in Example 5 was used in thisexample.

Chemicals—

Penicillin, streptomycin, Dulbecco's modified Eagle's medium (DMEM) wasfrom Mediatech (Herndon, Va.) and 10% FBS-HI (fetal bovine serum-heatinactivated) were obtained from Mediatech and Hyclone (Logan, Utah). Thecommercial supercritical CO₂ extracts of N. sativa produced in Example 1were used as the test materials. Unless noted, all other standardreagents were purchased from Sigma (St. Louis, Mo.).

Cell Culture—

Mouse C2C12 myoblasts were obtained from American Type CultureCollection (Manassas, Va.), and were maintained in Dulbecco's modifiedEagle's medium (DMEM) supplemented with 10% fetal bovine serum at 37° C.under a humidified atmosphere of 5% CO₂.

C2C12 cells were seeded at an initial density of 6×10⁴ cells/cm² in24-well plates. For two days, the cells were allowed grow to reachconfluence. Following confluence, the cells were forced to differentiateinto myocytes by culturing in DMEM supplemented with 2% horse serum forseven days.

Treatment—

On Day 8 to 10 post differentiation, C2C12 myocytes were incubated inserum-free DMEM plus 0.5% BSA (bovine serum albumin) for three hours.Next, AICAR (Cell Signal, Danvers, Mass.) was dissolved in phosphatebuffered saline (PBS) and added to the culture medium to achieveconcentrations of 1 mM. Test materials NS8, NS9 and NS10 were added inDMSO to achieve a final concentration of 25 μg test material/mL and onepercent DM SO. After 30 minutes at 37° C., cell lysates were preparedfor determination of activated AMPK.

Measuring Activated AMPKα—

pT¹⁷²-AMPK was quantified using the Biosource AMPK Immunoassay Kit(Camarillo, Calif.) without modification. Protein content of the celllysates was determined with the Active Motif fluorescent protein assayreagent (Carlsbad Calif., Hoefelschweiger, B. K., Duerkop, A., andWolfbeis, O. S, Novel type of general protein assay using a chromogenicand fluorogenic amine-reactive probe. Anal Biochem 2005, 344, 122-9). APackard Fluorocount spectrofluorometer (Model#BF 10000, Meridan, Conn.)was used for protein determination and a MEL312e BIO-KINETICS READER(Bio-Tek Instruments, Winooski, Vt.) was used for quantification ofpT¹⁷²-AMPK.

Calculation of Relative Activation of AMPK—

pT¹⁷²-AMPK was computed per mg lysate protein and then normalized to thedimethyl sulfoxide (DMSO) negative controls. For statisticalcomparisons, 95% confidence intervals were computed (Excel, Microsoft,Redman, Wash.).

Results—

Over ten independent assays, 1 mM AICAR increased pT¹⁷²-AMPK an averageof 1.67-fold (95% CI=1.26-2.21) in C2C12 myocytes relative to the DMSOnegative controls. In three independent assays, NS8 and NS9 fractionsactivated myocyte AMPK, while NS10 had no effect (FIG. 11). The AICARpositive control induced a 1.45-fold activation in assays run with N.sativa supercritical CO₂ extracts. In studies of direct comparisons, 25μg NS9/mL was 14% more active (p<0.05) than 1 mM AICAR in activatingAMPK.

Example 8 A Select Supercritical Fluid Extract of Nigella sativaOvercomes Adaptive Thermogenesis in Humans Resulting in Weight Loss

Objective—

The objective of this example was to assess the effect of supercriticalCO₂ extract NS9 prepared using the commercial-scale process described inExample 1 on adaptive thermogenesis in an individual involved in amoderate aerobic exercise regimen that initially produced weight loss,but was no longer effective.

Test Material—

Commercial scale NS9 as described in Example 1 containing 39% TQ wasused as the test material.

Methods—

A sixty-one year old male, who had been involved in a moderate, aerobicexercise program for a period of approximately one year, was directed totake two capsules of NS9 three times per day. The individual was furtherinstructed not to change his exercise regimen or dietary habits. Hisbody weight and blood pressure were recorded twice daily—within one hourof awakening in the morning and in the late afternoon for six monthsprior to the study and during the week of the study. The 99% confidenceinterval, computed from the previous six-month variation in day-to-daybody weight for AM and PM weighing, was used to assess significant(p<0.01) decreases, respectively, in AM or PM body weight.

Results—

Although the individual had maintained a stable weight over the previous12 months, during the week in which his regimen included 40 mg NS9/kghis body weight decreased approximately three percent or six pounds(FIG. 12).

Conclusion—

The incorporation of NS9, a supercritical CO₂ extract prepared fromcommercial-scale quantities of powdered N. sativa seeds, into theexercise and dietary regimen of an individual experiencing a plateau ofweight loss (adaptive thermogenesis) overcame adaptive thermogenesis tofacilitate further weight loss.

Example 9 Commercial Supercritical Fluid Extracts of Nigella sativaAttenuate LPS/Oxidant-Mediated Loss of Transepithelial ElectricalResistance in Caco-2 Intestinal Epithelial Cells More Effectively ThanThymoquinone

Objective—

The objective of this experiment was to assess the effect of thecommercial-scale supercritical CO₂ extracts of N. sativa produced inExample 1 on the loss of transepithelial electrical resistance in Caco-2monolayers induced by a cytokine/prooxidant stimulus.

Caco-2 Cells—

The protocol for growing and differentiation the human Caco-2 colonandenocarcinoma cells was a modification of Protocol 3 as described byYamashita et al. [Yamashita, S., Konishi, K., Yamazaki, Y., Taki, Y.,Sakane, T., Sezaki, H., and Furuyama, Y. 2002) New and better protocolsfor a short-term Caco-2 cell culture system, J Pharm Sci 91, 669-679].Caco-2 Human Colon Adenocarcinoma cells were obtained from ATCC(Rockville, Md.; catalog #HTB-37) and maintained in a growth media: DMEM(Dulbecco's Modified Eagles Medium lx) containing L-glutamine, glucose;Cellgro catalog #35-010CV with the following additions: (1) Penicillin(5,000 IU/mL)/Streptomycin (5,000 μg/mL) Cellgro catalog #30-001C1, (2)10% FBS—Fetal Bovine Serum, Characterized; Hyclone catalog #SH30071.3,and (3) 1% NEAA—Non Essential Amino Acids; Cellgro catalog #25-025-CI;cells were cultured at 37 C in a humidified air-5% CO₂ atmosphere inT175 flasks.

Differentiation of Caco-2 Cells to Intestinal Epithelial Cells—

BIOCOAT^(R) HTS Caco-2 Assay System kits (Becton Dickinson, N.J.;catalog #354801) consisting of 24-well fibrillar collagen coated insertsand feeder trays were used in all experiments and plated as follows.Media was removed from the T175 flasks and cells were washed with 10 mLsPBS (phosphate buffered saline 1× without Ca⁺⁺, without Mg⁺⁺; Cellgro#25-053-CI). PBS was removed and 5 mL of Trypsin/EDTA (lx, 0.25%Trypsin/2.21 mM EDTA in HBSS; Cellgro #25-053-CI) were added to theflask and the flask placed at 37 C until cells were visibly floating,approximately 3-5 minutes. Five mL of growth media were added to theflask to neutralize the trypsin. This solution was then transferred to asterile 50 mL tube. Eight μL were sampled and placed in a hemocytometerfor counting under a microscope. Five-hundred μL of cell solution wereplaced in the upper chamber of each insert so that the final density perwell was at least 6.6×10⁵ cells/cm². Thirty-five mL of growth media werethen added to the feeder tray and plate incubated at 37 C with 5% CO₂and 100% humidity for 20-24 hours. At this time, media were removed fromthe inserts by decanting and from the feeder tray by aspiration. Mediawere then added for the cell differentiation phase; Entero-STIM™ Mediumwas prepared as per assay kit instructions (Becton Dickinson, N.J.;catalog #354801), 500 μL to the upper chamber of each insert and 35 mLadded to the feeder tray.

This medium was refreshed after 48 hours. The following day the plateswere prepared for treatment. Media were removed from the upper chamberof the inserts so that the final volume was 300 mL. The insert plate wasremoved from the feeder tray and placed directly on a 24-well plate. OnemL of Entero-STIM™ Medium was added to each well.

Test materials were solubilized or suspended by sonication for 5 minutesin DMSO as a 500× stock and added to top wells in 0.6 μL to achieve thetabulated concentrations. DMSO was added to both negative and positivecontrols at the same 0.1% concentration as the test wells. Testmaterials remained on the monolayers for 1 hour, at which time allpositive and test cells were treated with 10 uM H₂O₂ and 50 μg/mLlipopolysaccharide (LPS, Sigma, St. Louis).

Transepithelial Electrical Resistance Assay—

Baseline transepithelial electrical resistance (TEER) measurements ofthe Caco-2 monolayers were made using a Millicell^(R)-ERS system(Millipore Corporation, Bedford, Mass.). Measurements of TEER were made1, 2 and 3 hours post treatment.

Data Analysis—

After TEER values were normalized to their zero-hour control, thepercent change from the positive LPS/H₂O₂ control at three hours wastabulated for each fraction. Ninety-five percent confidence intervals(CI) were computed for the positive control, which was set to 100(Excel, Microsoft, Redmond, Wash.). Values were considered significantlydifferent from the positive control if the mean of the four test wellsof the samples fell outside the 95% CI (p<0.05).

TABLE 6 Effect of supercritical CO₂ extracts of Nigella sativa onLPS/oxidant-mediated decrease of transepithelial electrical resistancein Caco-2 cells Test Concentration Relative Loss Treatment [μg TQequvalents/mL] of TEER LPS/H₂O₂ — 100 ± 7.41 Thymoquinone 5.0 111(Sigma) NS8 0.11 79.8* NS9 0.99 117 NS10 0.012 99.4 NSSpent#1 5 μgSpent#1/mL 99.6 NSSpent#2 0.0005 85.8* **Significantly less thanLPS/H₂O₂ positive control (p < 0.05)

Results—

TQ, NS9, NS10, and NSSpent#1 had no effect on the LPS/H₂O₂-mediated lossof TEER in Caco-2 monolayers (Table 6). NS8 and NSSpent#2, however,reduced the relative LPS/H₂O₂-stimulated loss of TEER, respectively, 21and 14 percent.

Conclusions—

Unexpectedly, the degree to which the extracts attenuated LPS/H₂O₂stimulation in differentiated Caco-2 monolayers was out of proportion tothe TQ content of all extracts.

Example 10 Supercritical Fluid Extracts of Nigella sativa Attenuatetrans-10,cis12-Conjugated Linoleic Acid Isomer Loss of TransepithelialElectrical Resistance in Caco-2 Intestinal Epithelial Cells

Background—

Several clinical studies have shown that HIV-1 infection is associatedwith increased permeability of the intestinal tract as evidenced byHIV-induced impairment of mucosal barriers. Exposure to HIV-1 candirectly breach the integrity of the mucosal epithelial barrier, andallow translocation of virus and bacteria to impair the gastrointestinalmucosal barrier contributing to further progression of the HIVinfection. Additionally, protease inhibitors (PI's) and reversetranscriptase drugs, components of highly active antiretroviral therapy(HAART) for treating human acquired immunodeficiency syndrome (AIDS),have been limited by undesirable side-effects, such as diarrhea and lossof intestinal membrane integrity (leaky gut syndrome, LGS). Dietarycomponents such as conjugated linoleic acid, suggested for use toovercome hyperlipidemia or lipodystrophy associated with HAART, may alsocontribute to LGS as the t10-CLA has been shown to negatively affectTEER in Caco-2 cells.

Objective—

The objective of this experiment was to assess the effect of thecommercial-scale supercritical CO₂ extracts of N. sativa produced inExample 1 on the loss of transepithelial electrical resistance in Caco-2monolayers induced by t10-CLA.

Methods—

Experimental methods and data analysis for assessing the loss of TEER int10-CLA stimulated Caco-2 cells were as described in the previousexample with the exception that 50 μM t10-CLA was used to induce TEERloss in place of the LPS/H₂O₂ positive control.

Results—

All supercritical CO₂ extracts of N. sativa as well as TQ inhibited lossof TEER in t10-CLA-stimulated Caco-2 cells indicating the ability toreduce intestinal monolayer disruption in response to a pro-inflammatorystimulus (Table 7). Spent#1 the extracted powdered seed material had noeffect. Unexpectedly, the degree to which the extracts attenuatedt10-CLA stimulation in differentiated Caco-2 monolayers was out ofproportion to the TQ content of all extracts.

TABLE 7 Effect of supercritical CO₂ extracts of Nigella sativa onLPS/oxidant- mediated decrease of transepithelial electrical resistancein Caco-2 Test Concentration Relative Loss Treatment [μg TQequvalents/mL] of TEER Solvent Only — 0.00* t10-CLA 50 μM 100 ± 5.81Thymoquinone 5.0 44.5* (Sigma) NS8 0.11 13.9* NS9 0.99 31.53* NS10 0.01225.1* NSSpent#1 5 μg Spent#1/mL 95.8 NSSpent#2 0.0005 0.00**Significantly less than t10-CLA 50 μM positive control (p < 0.05)

Example 11 Supercritical Fluid Extracts of Nigella sativa Attenuatetrans-10, cis12-Conjugated Linoleic Acid Isomer Mediated Inflammation in3T3-L1 Adipocytes

Background—

Inflammation plays a role in t10-CLA isomer mediated insulin resistancein adipocytes. One manifestation of this inflammatory response isincreased IL-6 and decreased adiponectin secretion by the adipocyte.

Objective—

The objective of this experiment was to assess the effect of thecommercial-scale supercritical CO₂ extracts of N. sativa produced inExample 1 on the increase in IL-6 secretion and decreased adiponectinsecretion in 3T3-L1 adipocytes induced by the t10-CLA isomer.

The Model—

The 3T3-L1 murine fibroblast model as used in Example 4 was used in thisexperiments.

Test Materials—

Commercial-scale supercritical CO₂ extracts of N. sativa produced inExample 1 were used as test materials. Powdered t10-CLA was providedfrom Lipid Nutrition (Wormerveer, The Netherlands).

Treatment—

Test materials were added four hours prior to the addition of t10-CLA ata concentration of 50 μM. Following overnight incubation, thesupernatant media were sampled for determination of IL-6 andadiponectin.

Interleukin-6 Assay—

The IL-6 secreted into the medium in response to TNFα stimulation wasquantified using the Quantikine® Mouse IL-6 Immunoassay kit with nomodifications (R&D Systems, Minneapolis, Minn.). Information supplied bythe manufacturer indicated that recovery of IL-6 spiked in mouse cellculture media averaged 99% with a 1:2 dilution and the minimumdetectable IL-6 concentration ranged from 1.3 to 1.8 pg/mL. Allsupernatant media samples were diluted 1:30 for quantification.

Adiponectin Assay—

The adiponectin secreted into the medium was quantified using the MouseAdiponectin Quantikine® Immunoassay kit with no modifications (R&DSystems, Minneapolis, Minn.). Information supplied by the manufacturerindicated that recovery of adiponectin spiked in mouse cell culturemedia averaged 103% and the minimum detectable adiponectin concentrationranged from 0.001 to 0.007 ng/ml.

Statistical Calculations and Interpretation—

Test materials and were assayed in duplicate, while solvent controlswere replicated eight times. IL-6 and adiponectin secretion wererepresented relative to the IL-6 and adiponectin secretion of thet10-CLA only controls as the IL-6 and adiponectin index and differencesamong the means were analyzed by the student's t-test assuming a fivepercent probability of a type I error (Excel; Microsoft, Redmond,Wash.).

Results—

Treatment with 50 μM t10-CLA induced a 8-fold increase in IL-6 secretionand 65 percent reduction in adiponectin secretion relative to controls(Table 8). All supercritical CO₂ extracts of N. sativa as well as TQinhibited IL-6 secretion in t10-CLA-stimulated adipocytes indicating theability to reduce secretion of inflammatory cytokines in response to apro-inflammatory stimulus. Similarly, the extracts and TQ attenuatedt10-CLA-stimulated decrease of adiponectin secretion. Spent#1 theextracted seed material had no effect. Surprisingly, the degree to whichthe extracts affected t10-CLA stimulation in 3T3-L1 adipocytes was outof proportion to the TQ content of all extracts.

TABLE 8 Effect of supercritical CO₂ extracts of Nigella sativa ont10-CLA-mediated inflammation in 3T3-L1 Adipocytes Test ConcentrationIL-6 Adiponectin Treatment [μg TQ equvalents/mL] Index Index SolventOnly —  12.5 222  t10-CLA 50 μM 100 ± 11 100 ± 14 Thymoquinone 5.0 75*128* (Sigma) NS8 0.11 59* 132* NS9 0.99 17* 188* NS10 0.012 69* 166*NSSpent#1 5 μg Spent#1/mL 98  109  NSSpent#2 0.0005 54* 143**Significantly less than t10-CLA 50 μM positive control (p < 0.05)

The attenuation of IL-6 secretion and inhibition of adiponectinsecretion of adipocytes as demonstrated in this example underscores thepotential of these novel supercritical CO₂ extracts of N. sativa toovercome the diabetogenic effects of t10-CLA. Such extracts would beuseful in combination with CLA mixed isomers containing t10-CLA toincrease efficacy for weight loss, metabolic syndrome or type 2diabetes.

Thus, among the various formulations taught there have been disclosedfour novel supercritical CO₂ extracts of N. sativa resulting fromproduction of commercial scale quantities of powdered seed containingabout 0.01 to about 39 (% w/w) TQ and produced under specific conditionsof temperature, pressure and time of extraction. Methods for theproduction of these formulations and uses have been described. It willbe readily apparent to those skilled in the art, however, that variouschanges and modifications of an obvious nature may be made withoutdeparting from the spirit of the invention, and all such changes andmodifications are considered to fall within the scope of the inventionas defined by the appended claims. Such changes and modifications wouldinclude, but not be limited to, the incipient ingredients added toaffect the capsule, tablet, powder, lotion, food or bar manufacturingprocess as well as vitamins, flavorings and carriers. Other such changesor modifications would include the use of herbs or other botanicalproducts containing the combinations of the preferred embodimentsdisclosed above. Many additional modifications and variations of theembodiments described herein may be made without departing from thescope, as is apparent to those skilled in the art. The specificembodiments described herein are offered by way of example only.

1. A composition for adipocyte modification for the treatment of metabolic disorders in a subject in need thereof, said composition comprising a therapeutically effective amount of a pharmaceutical composition comprising a therapeutically effective amount of a pharmaceutically acceptable botanical product wherein the botanical product is a compound or extract derived from the commercial-scale, supercritical fluid, oil extract composition from seeds of Nigella sativa derived by: a. grinding and sieving the seed of Nigella sativa to a fine powder of about 20 to 30 mesh; b. extracting quantities of the ground seeds with supercritical CO₂ at about 140 bar, at about 50° C. for about 30 minutes and collecting the oil fraction as the first extract composition (C1) obtained; c. setting aside C1 and continuing the extraction of the ground Nigella sativa seeds at about 140 bar, at about 50° C. for about 120 minutes and collecting the oil fraction as the second extract composition obtained (C2); d. setting aside C2 and continuing the extraction of the ground Nigella sativa seeds by increasing the pressure to about 300 bar, the temperature to about 60° C. and collecting the oil extract for about an additional 180 minutes as the third extract composition (C3) obtained; e. formulating a fourth extract composition (C4) by mixing the extracted, spent Nigella sativa powdered seeds with the third extract composition (C3) in a ratio of about 24:1.
 2. The method according to claim 1, wherein the resulting oil extract Composition C2 has a thymoquinone concentration of about 39%.
 3. The method according to claim 1, wherein the resulting oil extract composition C2 has a 2,2-diphenyl-1-picrylhydrazyl free radical quenching activity of about 150 μmol Trolox equivalents/g thymoquinone. 4-15. (canceled) 